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The salivary glands and saliva of Anopheles gambiae asan essential step in the Plasmodium life cycle: a global

proteomic study.Valérie Choumet, Annick Carmi-Leroy, Christine Laurent, Pascal Lenormand,

Jean-Claude Rousselle, Abdelkader Namane, Charles Roth, Paul T Brey

To cite this version:Valérie Choumet, Annick Carmi-Leroy, Christine Laurent, Pascal Lenormand, Jean-Claude Rousselle,et al.. The salivary glands and saliva of Anopheles gambiae as an essential step in the Plasmod-ium life cycle: a global proteomic study.. Proteomics, Wiley-VCH Verlag, 2007, 7 (18), pp.3384-94.�10.1002/pmic.200700334�. �pasteur-00527461�

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For Peer ReviewThe salivary glands and saliva of Anopheles gambiae as an essential step

in the Plasmodium life cycle: a global proteomics study

Journal: PROTEOMICS Manuscript ID: draft

Wiley - Manuscript type: Research Article Date Submitted by the

Author: n/a

Complete List of Authors: Choumet, Valerie; Institut Pasteur, Biochimie et Biologie Moleculaire des Insectes Carmi, Annick; Institut Pasteur, Biochimie et Biologie Moleculaire des Insectes Laurent, Christine; Institut Pasteur, Plate-Forme de protéomique Lenormand, Pascal; Institut Pasteur, Plate-Forme de protéomique Rousselle, Jean-Claude; Institut Pasteur, Plate-Forme de Protéomique Namane, Abdelkader; Institut Pasteur, Plate-Forme de protéomiqueRoth, Charles; Institut Pasteur, Biochimie et Biologie Moleculaire des Insectes Brey, Paul; Institut Pasteur, Biochimie et Biologie Moleculaire des Insectes

Key Words: Electrophoresis, Stage-specific proteins, Two-dimensional gel electrophoresis, Differential expression, Tandem mass spectrometry

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The salivary glands and saliva of Anopheles gambiae as an essential step in the

Plasmodium life cycle: a global proteomics study

Valérie Choumet1*, Annick Carmi1, Christine Laurent2, Pascal Lenormand2, Jean-Claude

Rousselle2, Abdelkader Namane2, Charles Roth1 and Paul T. Brey1

1 Unité de Biochimie et de Biologie Moléculaire des Insectes, Institut Pasteur, 28 rue du Dr

Roux, 75724 Paris cedex 15

2 Plate-forme de protéomique, Institut Pasteur, 28 rue du Dr Roux, 75724 Paris cedex 15

* to whom correspondance should be addressed: tel: 33145688630; fax: 33140613471; email:

[email protected]

Keywords: Anopheles gambiae, mosquito, Plasmodium berghei, proteomics, salivary gland

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SUMMARY

Proteins synthesized in the salivary glands of the Anopheles gambiae mosquito are thought to be

important in the life cycle of the malaria parasite Plasmodium. To describe Anopheles gambiae

salivary gland and saliva contents, we combined several techniques: 1-DE, 2-DE and LC

MS/MS. This study has identified five saliva proteins and 122 more proteins from the salivary

glands, including the first proteomic description for 89 of these salivary gland proteins. Since the

invasion and sporozoite maturation take place during the process of salivary glands ageing, the

effect of salivary gland age on salivary component composition was examined. LC MS/MS

profiling of young versus old salivary gland proteomes suggests that there is an over-

representation of proteins involved in signalling and proteins related to the immune response in

the proteins from older mosquitoes. iTRAQ labelling was used for a comparative proteomic

analysis of salivary gland samples from infected or Plasmodium berghei-free mosquitoes. The

expression levels of five secreted proteins were altered when the parasite was present. These

observations will serve as a basis for future work concerning the possible role of these proteins in

the interaction between A. gambiae, Plasmodium and the mammalian host.

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INTRODUCTION

Malaria is a parasitic disease that affects 200 million people worldwide and causes 1.5 to 2.7

million deaths per year. Of the 300-500 million clinical cases annually, nearly 90% are in the

sub-Saharan countries of Africa where the malaria parasite, Plasmodium falciparum, is primarily

transmitted by the mosquito Anopheles gambiae. The increasing resistance of the parasite to

inexpensive drugs and the resistance of mosquitoes to insecticides have created an urgent need

for innovative methods that block parasite transmission during its development within the insect.

The Anopheles mosquito not only carries the parasite from infected to uninfected people, but also

plays a vital role in the parasite life cycle [1]. Mosquito saliva and salivary glands are central to

the interaction between parasite, vector and mammalian host. Sporozoite maturation in the

mosquito salivary glands before its transmission to vertebrates is a key stage for the effective

transmission to humans since it increases the sporozoite’s ability to infect vertebrate hepatocytes

[2]. Additionally, sporozoites are injected into the vertebrate skin with nanolitre volumes of

saliva, a complex biologically active solution, which, in addition to other activities, serves as the

“transmission fluid” for the malaria parasite.

The salivary glands and their diversified protein contents are essential for overcoming the

challenges posed by the host: pain and itch responses, immune defences and haemostasis [3].

There is convincing evidence that the pharmacological activity of arthropod saliva affects

pathogen transmission. Salivary gland lysate from the sand fly Lutzomia longipalpis facilitates

the infection of mice by the protozoan parasite Leishmania major [4, 5]. However, there has been

little work on the role of mosquito salivary gland proteins in promoting infection of Plasmodium

species in vertebrate hosts. During the last 3 years, there have been several studies on the

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transcriptome and the proteome of salivary glands of arthropod vector saliva [6-11]. Kalume et

al. [12] identified 67 proteins from Anopheles gambiae salivary glands, an initial step towards the

cataloging of the hundreds of proteins and peptides in the salivary proteome. However, no

attempts have been made to study the proteome of Anopheles gambiae saliva in the presence of

malaria parasite.

This communication presents an expanded investigation of saliva and salivary proteins in blood-

fed A. gambiae mosquitoes determined by several proteomics approaches. These techniques

ensured good coverage of salivary gland proteins of varied pIs and molecular weights. The

iTRAQ labelling technology was used to quantitate differences in the proteomes of Plasmodium

berghei-infected and non-infected A. gambiae salivary glands.

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MATERIALS AND METHODS

Reagents

Mosquitoes

Yaounde strain adult A. gambiae females were reared in insect rooms at 26±0.5°C, 70% relative

humidity, with a 16h/8h light : dark photoperiod. The adult female mosquitoes used in these

experiments were either aged between 5 and 8 days or between 18 and 21 days and had blood

meals 3 to 5 days after emergence. Plasmodium berghei NK65 strain parasites, transformed to

express GFP at the sporozoite stage, were injected into mice by intraperitoneal injection; seven

days later, female mosquitoes aged 2-3 days were fed on the infected mice. All mosquitoes were

maintained on a diet of 10% Karo syrup solution. Salivary glands from either 5-8 day old or at

18-21 day old mosquitoes were dissected in 150 mM NaCl with protease inhibitors (Complete,

Roche Diagnostics, Manheim, Germany ) at 4°C and stored at –80°C. Saliva was collected using

artificial feeders. After lyophilisation, saliva components were re-suspended in water and stored

at -80°C.

Salivary gland extract preparation

Salivary glands were disrupted by ultrasound (Cup horn, Sonics & Materials Inc., Newton, CT,

USA) for 20 min at maximum amplitude. Salivary gland homogenates were then centrifuged for

30 min at 130,000g and protein was quantified using the BCATM protein assay (Pierce, Rockville,

IL, USA). Aliquots of salivary gland extracts were stored at -80°C until use.

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SDS PAGE

SG samples of 10 µg or 36 µg of salivary gland were dissolved in Laemmli sample buffer, and

boiled for 5 min. After centrifugation (14000rpm, 10 min), 20 µl samples were loaded onto a

12% acrylamide, 1mm-thick SDS PAGE Bis-Tris minigel, and subjected to electrophoresis on a

Novex apparatus (Invitrogen, Carlsbad, CA, USA). Protein molecular weight markers (Precision

Plus Protein standard all blue, Bio-Rad, Hercules, CA, USA) were run on the same gel. The gel

was stained with Bio-SafeTM Coomassie (Bio-Rad) or silver nitrate (PlusOneTM, GE Healthcare,

Uppsala, Sueden). Two methods were used to isolate proteins from the gel for mass

spectrometry. One method consisted of cutting out all bands visible after Coomassie or silver

staining. The other method consisted of cutting the gel into 1 mm-thick slices. The plugs obtained

were analyzed by mass spectrometry.

2-DE

Samples of salivary gland supernatant, corresponding to 50 or 120 µg of protein, were used for 2-

D gel analysis. To improve 2-D gel profiles, samples were treated using a ReadyPrep 2-D

Cleanup kit (Bio-Rad, Hercules, CA, USA). The pellet recovered after the last centrifugation step

was dissolved in 15 mM NaCl, 0.5% SDS (final concentration), and 2% Triton X100 (final

concentration). The sample was heated at 95°C for 3 min, flash-frozen in liquid nitrogen and

lyophilized. The lyophilized material was dissolved in 2-DE sample buffer (7M urea, 2M

thiourea, 4% CHAPS, 150 mM DTT, and 2% ampholytes).

SG samples (30µl) were loaded onto IEF 18cm gels containing ampholines of pH ranging from 4

to 8 (Bio-Rad), and run for 20000 Vhrs. The second dimension was carried out on 12.5%

acrylamide 22cm slab gels. Resolved proteins were detected by SYPRO®Ruby (Invitrogen). For

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each type of salivary gland extract (young blood-fed, 21 day blood-fed, infected), at least three

independent sample preparations were used, and at least three independent gel analyses were

carried out.

Mass spectrometry

MALDI-TOF-MS and database searches

Mass spectrometry was performed using a MALDI-TOF instrument (Voyager-DE-STR, Applied

Biosystems, Framingham, MA) operated in positive ion reflector mode. Sample preparation for

in-gel digestion was carried out as described previously [13]. Bands and spots of interest were cut

out using the Investigator ProPic robot (Genomic Solutions). Plugs were washed with 100 mM

ammonium bicarbonate (Sigma) and proteins reduced with 10 mM 1,4-dithiothreitol (Sigma,

Saint-Louis, MO, USA), S-alkylated with 55 mM iodoacetamide (Sigma) and in-gel digested at

37°C for 4 hours with modified porcine trypsin (Promega) using the Investigator ProGest robot

(Genomic Solutions, Ann Arbor, MI, USA). Peptide mixtures were desalted on ZipTip C18

(Millipore) and directly eluted onto the Maldi target using the Investigator ProMS robot

(Genomic Solutions). The elution solvent consisted of a six-fold dilution of a saturated solution

of CHCA (10mg/ml, Sigma) in 70% ACN (J.T. Baker) containing 0.1% TFA (Sigma). Each mass

spectrum (700-3000 m/z) was acquired in automatic mode (12 sub-spectra of 50 laser shots were

accumulated). Trypsin autolysis peptides were used as internal calibratants (fragment 108-115:

[M+H]+= 842.5100 and fragment 58-77: [M+H]+= 2211.1046). A local copy of MS-FIT 3.2

software, part of the Protein Prospector package (University of California, San Francisco) was

used to search the NCBI or Anopheles Ensembl databases. Search parameters were set as follows:

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only monoisotopic masses were used, a maximum peptide mass error of 50 ppm was allowed and

one incomplete cleavage per peptide and a possible oxidation of methionine were considered.

Moreover, no restrictions on Mr or pI were made, and a minimum of four matching peptides

covering a minimum of 15% of the protein sequence, were required for protein identification. If

necessary, MALDI-TOF-PSD experiments were carried out to reach protein identifications using

MS-TAG software (part of Protein Prospector package).

LC MS/MS

Protein digestion before identification by LC MSMS

Proteins were reduced, alkylated with 10 mM iodoacetamide, and digested with porcine trypsin

(ratio 1:100) overnight at 37°C. The trypsin digests were desalted with C18 tips (OMIX, Varian),

and stored at -80°C before LC MS/MS analysis.

LC MS/MS analysis

Prior to reverse phase nanobore liquid chromatography tandem mass spectrometry (nanoLC

MS/MS) analysis, samples were dissolved in Solvent A containing 5% acetonitrile and 0.1%

formic acid. The nanobore LC system was from LC Packings (Amsterdam, The Netherlands),

and consisted of a Famos autosampler and an Ultimate Nano LC system. It was interfaced with a

QqTOF mass spectrometer, QSTAR XL (AB/MDS Sciex, Foster City, CA), using a

nanoelectrospray source (Protana Engineering A/S, Odense, Denmark). Reverse phase LC was

performed using a PepMap column (75-µm inner diameter x 150-mm long, LC Packings, Dionex)

equilibrated with Solvant A. The peptides were eluted using a linear gradient of 5% to 40%

solvent B (95% acetonitrile, 5% H2O, and 0.1% formic acid) in 90 min with a flow rate of 200

nl/min. This binary gradient was used for protein identification and iTRAQ experiments. We

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operated the QSTAR XL mass spectrometer in an information-dependent-acquisition (IDA)

mode; each full MS scan was followed by two MS/MS scans where the two most abundant

peptide molecular ions were dynamically selected for CID, and dynamic exclusion was used to

prevent repetitive selection of the same ions within a preset time. Collision energies were set to

automatically adjust according to the charge state of the precursor ions.

iTRAQ Sample Preparation Procedure.

We denatured 40µg of each sample protein and blocked the cysteines as described in the iTRAQ

protocol (Applied Biosystems, Foster City, CA). Each sample was then digested with trypsin

solution overnight at 37 °C, and labelled with the iTRAQ tags as follows: non infected salivary

glands, iTRAQ114; infected salivary glands by P. falciparum, iTRAQ116 or iTRAQ 117. The

labelled samples were pooled and acidified for strong cation exchange (SCX) chromatography.

The eluted peptides were then lyophilised and stored at -81°C before analysis.

Database search and relative quantification

MS/MS data were analyzed using ProID protein identification software version 1.1 (AB/MDS

Sciex, Foster City, CA) using A. gambiae ORF database (Ensembl) [14]. In ProID, the peptide

tolerance and the MS/MS tolerance were set to 0.15 Da. We manually inspected the MS/MS

spectra to validate the identified peptides.

ProQUANT 1.1 (AB/MDS Sciex, Foster City, CA) and the A. gambiae ORF database (Ensembl)

were used to analyze data from the iTRAQ experiments. The confidence cut off was 95. The

tolerances set for peptide identification in ProQUANT searches were 0.15 Da for MS and 0.1 Da

for MS/MS. We manually validated all identifications. Relative protein quantification in iTRAQ

experiments was performed on the MS/MS scans and was the ratio of the areas under the peaks of

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iTRAQ reagent tags at 114, 116, and 117 Da. The quantification results were normalized using

the overall ratio obtained for all tagged peptide pairs in the sample.

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RESULTS AND DISCUSSION

Analysis of salivary gland, saliva and saliva components of 8 day-old blood-fed Anopheles

gambiae

One-dimensional electrophoresis

Salivary gland extracts

Two series of experiments were performed. In the first series, 12% SDS-PAGE gels were run

with 10 µg of protein extract obtained from salivary glands of 8 day-old females. After

Coomassie staining, protein bands were excised and the tryptic digests were analyzed by

MALDI-TOF mass spectrometry. In the second series, 12% SDS-PAGE gels were run with 36 µg

of protein extract. After Coomassie staining, 1 mm-thick plugs were cut from the gel (Figure 1,

supplementary Table 1). Protein identification was performed as described in Methods and

seventy percent of the bands were identified (Table 1).

Saliva

A total of 18 saliva samples each from 400 female 8 day-old blood-fed A. gambiae were

collected in water. After lyophilization, saliva components were resuspended in water and

analyzed by SDS-PAGE and stained with silver nitrate (Figure 2). The stained gel bands were cut

and analyzed by mass spectrometry. Five proteins were thereby identified (Table 1).

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Two-dimensional analysis

After 2-D gel electrophoresis of 120 µg of salivary gland proteins, the trypsin-digested spots

were analyzed by peptide mass fingerprinting, using Maldi-Tof, or by PSD Maldi-Tof. From the

total set of 204 spots (Figure 3), 29 proteins were identified and described (Table 1,

supplementary Table 2). MS identification showed that 37% of these proteins produced several

spots during electrophoresis. Spots at varying pIs were found for the putative 5’ nucleotidase

precursor in the 62-kDa region of the gel (spots 13 to 33 in Figure 3) as well as for the D7

precursor allergen AED A2 in the 30-kDa region (spots 114-119, 121-125) and for D7 related-4

protein precursor in the 16 kDa region (spots 171-176). The profile of the 30 kDa protein was of

particular interest with its intense spot at 32.5 kDa and a trail of spots with molecular weights

between 30 to 20 kDa (Figure 3). According to the Ensembl database (release 35), two forms of

the protein exist, including a long mature form of 24732.75 kDa (ensangp000000028522), and a

short mature form of 13786.59 kDa (ensangp00000022344); however, only the short form

remained in the Ensembl release 43. The proteomic data are consistent with a larger form of the

D7 precursor that is processed by proteolytic cleavage. Several other spots identified as being

secreted proteins had apparent Mr smaller than expected according to their genomic predicted Mr

in Ensembl (Table 1). This was the case for the 5’nucleotidase precursor protein that was

identified in spots of apparent molecular weights ranging from 62 kDa to 29 kDa (Table 1, Figure

3). To determine whether this range of sizes is due to an artifact that occurs before or during

sample preparation, 2-DE profiles of salivary gland extracts obtained after several freeze/thaw

cycles were examined. These profiles did not differ from those of extracts obtained after our

normal sonication and centrifugation procedure (data not shown). Additionally, the heating stage

was not responsible for proteolysis since the numbers of spots observed with heated salivary

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gland extracts and those not heated were similar (data not shown). These observations indicate

that several secreted proteins may present sequence divergence or be extensively processed

and/or post-translationaly modified in A. gambiae salivary glands. This idea is supported by the

following points: 1) several proteins were only identified after post source decay; 2) an extensive

processing of the human saliva proteome has been described [15].

Identification of salivary gland components using LC MS/MS

LC MS/MS is an alternative strategy for large-scale protein identification that bypasses the initial

protein separation step. It consists of enzymatic cleavage of a complex protein mixture and

separation of the resulting peptides by chromatography before tandem mass spectrometry

identification. This gel-free strategy has worked for large-scale protein identification of several

biochemical systems [16].

Using this system, 30 proteins were identified with confidence (ProtScore cutoff > to 95%). Of

these 30 proteins, 15 proteins (50 %) were matched with two or more peptides and the other 50%

were identified by a single peptide hit (Table 1). Using this technique, we were able to confirm

that there is a problem in the ensangp00000022344 annotation corresponding to our 30 kDa

protein, since only two of the five peptide sequences identified by LC MS/MS were present in the

current ensangp00000022344 sequence (Ensembl release 43).

Proteome coverage of 8 day-old blood-fed Anopheles gambiae salivary gland

Together, the three technologies characterised 55 different proteins, four of which

(ensangp00000028522, ensangp00000026134, ensangp00000027538, ensangp00000015472) are

no longer present in the latest genome annotation (Ensembl release 43). LC MS/MS and 2-DE-

MS identified a similar number of proteins and both appear more effective than 1-DE-MS. Thirty

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percent of the proteins identified are secreted. Ensangp00000013568, which is predicted to have

aspartic-protease activity, is one of the newly identified proteins. Blast analysis has shown that

this protein has 88% sequence identity with protein AAEL006169-PA in the Aedes aegypti

genome and is also similar to cathepsin D enzymes of other insects such as Drosophila

melanogaster and Bombyx mori. Insect cathepsins D have been shown to be involved in

metamorphosis [17] and their levels are modulated in pathogen-infected insect tissues [18].

Analysis of salivary gland components of 21 day-old blood-fed Anopheles gambiae salivary

glands and comparison of salivary gland components from young (8 days) and old (21 days)

blood-fed female mosquitoes

Plasmodium berghei development in A. gambiae takes about 14 days from the infective

bloodmeal until the parasite is ready to infect its mammalian host. Thus, the proteomic profile of

salivary glands may be affected by ageing. To identify the molecular changes that may occur in

salivary gland cells, the proteomic profile of 21 day-old female salivary glands was analyzed by

LC MS/MS (Table 2). A total of 41 different proteins were characterised (Table 2). Nineteen of

these proteins were described at a proteome level for the first time. Ensangp00000029528

(apolipoprotein D precursor), a protein identified as an infection-responsive protein in the

Anopheles midgut [19], was one of these proteins described for the first time. iRNA silencing of

the midgut transcript encoding APOD resulted in increased Plasmodium levels. Also among the

newly identified intracellular proteins, ensangp00000029324 deserves particular attention. This

protein belongs to the family of α2-macroglobulins and has 64% sequence identity with TEP15

in a FASTA comparison. These thioester-containing proteins are protease inhibitors that can play

an important role in immune responses. Ensangp00000029324 has 39.42% sequence identity

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with a protein described in Ornithodoros moubata [20]. This O. moubata protein is expressed in

tick salivary glands, haemocytes and Malpighian tubules and its expression is enhanced in

response to a blood meal. Using gene expression screening for immune response genes in the A.

gambiae transcriptome, Oduol et al. [21] identified an α2-macroglobulin-related molecule that

responded strongly to malaria parasite infection. Thus, one could propose that

ensangp00000029324 is involved in the defence against pathogens such as parasites, bacteria or

fungi.

Using only the LC MS/MS identified proteins, the level of salivary components after 8 days was

compared to those after 21 days of salivary gland development. Their functions were compared

(Figure 4). The composition of young salivary glands was less diverse than that of older salivary

glands: i.e. fewer proteins could be identified in young salivary glands (29) than in older glands

(42). Eighteen proteins were common to both salivary gland extracts and most were secreted

proteins (30 kDa, apyrase, 5’ nucleotidase, D7 precursor allergen AED A2, D7r1, D7r2, D7r3

and D7r4, maltase precursor, peroxidase precursor, GSG6, GSG7, putative gVAG); among the

other identified proteins, three were glycolytic enzymes (phosphoglycerate mutase, malate

dehydrogenase, triosephosphate isomerase), one was an RNAse and another one was an actin-

binding protein (ensangp00000012938). All the secreted proteins identified in 8 day-old salivary

glands were also found in 21 day-old salivary glands, whereas 6 additional, secreted proteins (D7

r5, GSG5, lysozyme and the hypothetical proteins 8.8, 10 and 10.2 kDa) were specific to the 21

day-old salivary glands. Protein functions, including transcription, signalling and metabolism,

assigned to some of the housekeeping proteins that were found in 8 day-old salivary glands were

also identified in 21 day-old salivary glands, although a larger variety of proteins were associated

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with signalling and transcription regulation. Additionally, apolipoprotein D, lysozyme and α2-

macroglobulin, involved in the response to pathogens, were detected in 21 day-old salivary

glands, but were not detected by LC MS/MS in 8 day-old salivary glands. A partial list of age-

related mammalian protein variation from the study of ageing mammalian organs [22] includes

proteins involved in: (i) telomere repair, (ii) stress response, (iii) anti-oxidant defence, (iv)

nicotinamide deamination, (v) insulin/insulin-like growth factor-1 signalling,, (vi) histone

deacetylation, and (vii) regulation of the transcription of specific proteins, such as those involved

in pituitary development. Specific age related signatures in the transcriptome of Drosophila body

parts have also been investigated [23]. That study showed the presence of up-regulated mRNA

levels in the aged thorax, where salivary glands are located, for immune response genes, genes

linked to cellular morphogenesis as well as those for actin filament-based processes. Cellular

components of the endoplasmic reticulum and the proteasome complex were also over-

represented. Thus, our observations indicating an increased level of a subset of salivary gland

proteins is consistent with the transcriptional results observed in Drosophila. Interestingly,

proteins involved in lipid metabolisms were only identified in 21 day-old salivary glands. Lipids

are known to be important for parasite matabolism. Rosinski-Chupin et al. [11] showed that

genes involved in lipid metabolism were up-regulated by Plasmodium berghei. This observation

suggests that the maturation of sporozoites may require happening in ageing salivary glands.

Comparison of infected and non-infected salivary gland composition

iTRAQ, an isotope labelling approach, was used for the quantitative study of gene expression at

the proteome level. This approach is based on chemical isobaric tagging of the N-terminus of

peptides generated from trypsin digests of proteins isolated from cells or tissues in different

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states. The labelled samples are combined, fractionated together by strong cation exchange

chromatography and analysed by nanoLC mass spectrometry. The labelled peptides and hence

the corresponding proteins are then identified by database searching using the MS/MS data. The

fragmentation of the tag attached to the peptides generates a low molecular mass reporter ion

which is unique to the tag used. Comparison of the intensities of these reporter ions gives relative

protein quantification. Table 3 shows the list of proteins identified and quantified using iTRAQ.

Twelve identical, secreted proteins were found in uninfected and infected salivary glands during

three separate comparisons (Table 3). The ratios of reporter ion peaks of infected versus non-

infected salivary glands varied between 0.65 and 1.97. From the ratio values, it was deduced that

the expression of five of the proteins, was altered. The level of gVAG is increased two-fold in

infected salivary glands, whereas the levels of GSG6, apyrase, D7 related-1 protein precursor and

D7 precursor allergen AED A2 are decreased with ratios ranging from 0.67 to 0.77 for these

proteins (Table 3). The presence of pathogens in salivary glands has been reported to induce

modifications in insect behaviour and/or modifications in saliva composition. gVAG is a protein

of the antigen 5 family and it has similarities with the mammalian cysteine-rich secretory

proteins, vespid antigen 5 and plant-pathogenesis-related proteins [27]. The precise function of

these secreted proteins is unknown. The level of gVAG mRNA was shown to be increased in the

midgut of mosquitoes infected with Plasmodium falciparum compared to level in uninfected

midguts [19]. The silencing of this gene resulted in increased Plasmodium levels, suggesting that

gVAG is a defence-related protein [19]. We therefore expect a similar role of gVAG in A.

gambiae salivary glands.

The level of apyrase was reduced by a factor of 1.5 in P. berghei-infected A. gambiae salivary

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glands. Apyrase inhibits ADP-induced platelet aggregation and, therefore, affects blood-feeding.

The level of apyrase influences the probing time of Anopheles gambiae [28]. The reduction of

apyrase abundancy by 85% in salivary glands from A. gambiae due to the injection of double-

stranded apyrase encoding RNA was correlated with increased probing time. Decreased apyrase

levels in Plasmodium gallinaceum infected Aedes aegypti salivary glands caused an increase in

mosquito probing time [24]. An increase in probing time has also been observed for Anopheles

gambiae infected with Plasmodium falciparum [29]. Additionally, transcription of the apyrase

encoding gene appears to be repressed in P. berghei-infected A. gambiae mosquitoes [11]. Our

observation is, therefore, consistent with these data and we can expect an increase probing time

for P. berghei-infected A. gambiae.

The levels of D7 precursor allergen AED A2 and D7 related-1 protein precursor proteins were

decreased in infected salivary glands by a factor of 1.3 and 1.5 respectively. The D7 short

proteins bind serotonin with high affinity, as well as histamine and norepinephrine, thus

antagonizing the vasoconstrictor, platelet-aggregating, and pain-inducing level of these factors

[30]. The decreased production of D7 related-1 protein precursor may induce an increased local

inflammatory response to mosquito bites, thus modifying the immune response to the parasite.

Although we did not observe a change in D7 related-4 protein precursor protein levels in our

analysis, Rosinski-Chupin et al. [11] observed variable D7 related-4 protein precursor gene

expression using SAGE. The proteins D7 precursor allergen AED A2 and GSG6 have no known

function, thus we cannot anticipate the consequence of reducing their expression on parasite

development and transmission.

Table 3 also shows that the iTRAQ technique identified forty three proteins not observed using

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by LC MS/MS analysis of salivary gland extracts from insects of the same age. This result is

consistent with the previous observations that better fragmentation is obtained using this

technology, giving more peptides per protein and allowing the identification of less abundant

proteins [31, 32]. One of the newly identified proteins was a homolog of “translationally

controlled tumour protein” (Tctp). Tctp homologues have been described in mammals and in

many other species, including plants, earthworm, parasites, hydra and yeast [33-38]. They are

heat stable, calcium-binding proteins [39] and their expression is induced in response to various

stimuli within cells [38]. Tctps also bind haem, and tubulin [40]. Tctps induce the release of

histamine [41] and the secretion of interleukin-4 [42] from basophils. Despite having a ubiquitous

tissue distribution, multiple specific potencies [43] and highly conserved amino acid sequences,

their primary physiological role remains unclear [40]. The A. gambiae protein has the highest

identity scores with its Aedes albopictus and Aedes aegypti homologs (85%). An identity score of

44% was observed with the ticks Dermacentor and Ixodes salivary histamine-releasing factors

(HRF) [44, 45]. The tick HRF recombinant protein induced histamine secretion from a rat

basophilic leukaemic cell line, in a dose-dependent manner. We suggest that the Anopheles

gambiae Tctp homolog is present in saliva and contributes to the allergic inflammation associated

with the Anopheles gambiae bite. Thus, if it is similarly able to trigger cutaneous mast cell

histamine release, as observed with the Schistosoma mansoni Tctp homolog [46], the resulting

vasodilation could facilitate Plasmodium sporozoite migration into blood vessels.

Our data also identify Serpin 9 (ensangp00000016680) at the proteomic level for the first time.

Serpins are a very large family of serine protease inhibitors with various biological functions that

are found in all higher eukaryotes and viruses [47]. The mosquito genome contains 14 annotated

serpin genes, 10 of which are inhibitory protease substrates. Some of these serpins are involved

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in immune signal amplification cascades. Serpin 9 is involved in the arthropod immune response

and during Staphylococcus aureus infection, it is only induced late in infection [48]. In contrast,

during the Plasmodium life cycle in mosquitoes, serpin 9 is primarily activated when the midgut

epithelium is invaded by ookinetes [48]. However, a tag corresponding to Serpin 9 was identified

in the A. gambiae salivary gland using SAGE [11], but the level of this tag was not modified by

Plasmodium infection.

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CONCLUDING REMARKS

In this study, complementary proteomic approaches were used to catalogue 122 Anopheles

gambiae salivary gland proteins from blood-fed 8 day-old and 21 day-old females

(supplementary Table 3). The most acidic proteins identified were the 30 kDa protein (pI, 3.8)

and calmodulin (pI, 3.9) and the most basic proteins were retrovirus-related pol polyprotein (pI,

11.28) and ensangp00000015472 (pI, 10.38). The smallest proteins identified were hypothetical

8.8 kDa (Mr, 8.8 kDa) and retrovirus related pol polyprotein (Mr, 9.6 kDa) and the largest was

Ryanodin receptor 1 (Mr, 577.8 kDa). Our approach confirmed the presence of seven proteins

identified in earlier Ensembl annotations but not listed in the latest version (version 43). This

observation emphasizes the complementarity of proteomic and genomic approaches for accurate

genome annotation, an idea previously suggested by Kalume et al. [49].

LC MS/MS was clearly the most powerful technique (Figure 6A). iTRAQ labelling led to the

identification of 78 proteins, 39 of which were not identified by classical LC MS/MS, illustrating

the value of using the two technologies in parallel for maximum proteome coverage. The proteins

identified in this study were sorted into functional categories based on their annotations in the

database and the results are summarised in Figure 6B. A large proportion of the identified

proteins are involved in energy pathways, blood or sugar feeding, protein folding, modification

and in amino acid metabolism, but the largest group (37%) is composed of proteins with no

known function. The same situation is also encountered in the proteomic analysis of human

saliva [50]. In Anopheles gambiae, twenty five percent of the identified proteins are predicted to

have a signal sequence and are, therefore, putatively present in saliva. The largest category of

peptide sequences was that derived from secreted proteins, demonstrating that they are the most

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abundant proteins in salivary gland extracts. This observation is consistent with the findings of

Kalume et al. [12].

Seventy-five percent of the 122 proteins reported here are identified in an Anopheles gambiae

salivary gland proteomic study for the first time. Most of these newly identified proteins are

housekeeping proteins and only few, such as GSG5, GSG3, ensangp00000029324, serpin 9,

hypothetical 10 kDa and apolipoprotein D precursor, are secreted. The 2-D gel analysis suggests

that some secreted proteins, including 5’nucleotidase, D7 precursor allergen AED A2, D7

related-4 protein precursor and 30 kDa, are extensively processed, although the consequence of

such modifications on their activity is unknown. LC/MS-MS profiling of young versus old

salivary gland proteomes suggests that there is an over-representation of proteins involved in

signalling, proteins implied in carbohydrate and lipid metabolism and proteins related to the

immune response in older glands. As the invasion and the maturation of sporozoites occurs

during the ageing process of salivary glands, it would be interesting to know whether the age of

the salivary gland affects parasite transmission. Finally, we detected a change in the level of five

salivary proteins in the presence of Plasmodium berghei sporozoites. These observations will

serve as a basis for future work to determine the possible role of these proteins in the interaction

between A. gambiae, Plasmodium and the mammal host.

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ACKNOWLEDGEMENTS

We are grateful to Jean Sautereau and the CEPIA for mosquito rearing and infections. This work

was supported by a Grand Programme Horizontal grant from Institut Pasteur (GPH Anophèle)

and a CNRS “post-séquençage anophèle” grant.

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REFERENCES

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[28] Boisson, B., Jacques, J. C., Choumet, V., Martin, E., et al., FEBS Lett 2006, 580, 1988-1992. [29] Wekesa, J. W., Copeland, R. S., Mwangi, R. W., Am J Trop Med Hyg 1992, 47, 484-488. [30] Calvo, E., Mans, B. J., Andersen, J. F., Ribeiro, J. M., J Biol Chem 2006, 281, 1935-1942. [31] Hardt, M., Witkowska, H. E., Webb, S., Thomas, L. R., et al., Anal Chem 2005, 77, 4947-4954. [32] Aggarwal, K., Choe, L. H., Lee, K. H., Brief Funct Genomic Proteomic 2006, 5, 112-120. [33] Haghighat, N. G., Ruben, L., Mol Biochem Parasitol 1992, 51, 99-110. [34] Pay, A., Heberle-Bors, E., Hirt, H., Plant Mol Biol 1992, 19, 501-503. [35] Rasmussen, S. W., Yeast 1994, 10 Suppl A, S63-68. [36] Bini, L., Heid, H., Liberatori, S., Geier, G., et al., Electrophoresis 1997, 18, 557-562. [37] Bhisutthibhan, J., Pan, X. Q., Hossler, P. A., Walker, D. J., et al., J Biol Chem 1998, 273,16192-16198. [38] Sturzenbaum, S. R., Kille, P., Morgan, A. J., Biochim Biophys Acta 1998, 1398, 294-304. [39] Kim, M., Jung, Y., Lee, K., Kim, C., Arch Pharm Res 2000, 23, 633-636. [40] Gachet, Y., Tournier, S., Lee, M., Lazaris-Karatzas, A., et al., J Cell Sci 1999, 112 ( Pt 8),1257-1271. [41] MacDonald, S. M., Rafnar, T., Langdon, J., Lichtenstein, L. M., Science 1995, 269, 688-690. [42] Schroeder, J. T., Lichtenstein, L. M., MacDonald, S. M., J Exp Med 1996, 183, 1265-1270. [43] Bommer, U. A., Thiele, B. J., Int J Biochem Cell Biol 2004, 36, 379-385. [44] Mulenga, A., Azad, A. F., Exp Appl Acarol 2005, 37, 215-229. [45] Mulenga, A., Macaluso, K. R., Simser, J. A., Azad, A. F., Insect Biochem Mol Biol 2003, 33,911-919. [46] Rao, K. V., Chen, L., Gnanasekar, M., Ramaswamy, K., J Biol Chem 2002, 277, 31207-31213. [47] Irving, J. A., Pike, R. N., Lesk, A. M., Whisstock, J. C., Genome Res 2000, 10, 1845-1864. [48] Christophides, G. K., Zdobnov, E., Barillas-Mury, C., Birney, E., et al., Science 2002, 298,159-165. [49] Kalume, D. E., Peri, S., Reddy, R., Zhong, J., et al., BMC Genomics 2005, 6, 128.[50] Hu, S., Xie, Y., Ramachandran, P., Ogorzalek Loo, R. R., et al., Proteomics 2005, 5, 1714-1728. [51] Arca, B., Lombardo, F., Valenzuela, J. G., Francischetti, I. M., et al., J Exp Biol 2005, 208,3971-3986. [52] Hirtz, C., Chevalier, F., Centeno, D., Egea, J. C., et al., J Physiol Biochem 2005, 61, 469-480. [53] Calvo, E., deBianchi, A. G., James, A. A., Marinotti, O., Insect Biochem Mol Biol 2002, 32,1419-1427.

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LEGENDS TO FIGURES

Figure 1: SDS-PAGE of salivary gland extracts from 8 day-old blood-fed A. gambiae

Salivary components were separated by a 12% NU-PAGE Bis-Tris gel under denaturating and

reducing conditions. Molecular mass markers are shown on the left. After Coomassie staining,

the gel was cut into millimeter slices as indicated by the numbers on the right side of the figure.

The plugs obtained were analyzed by mass spectrometry as described in the Methods section.

Figure 2: SDS-PAGE of 8 day-old blood-fed A. gambiae saliva

Saliva was collected from 7200 females using artificial feeders. After lyophilisation, saliva

components were re-suspended in water and aliquots were analyzed by SDS-PAGE. Following

silver nitrate staining, the numbered protein bands were analyzed by mass spectrometry.

Figure 3: 2-DE analysis of salivary gland extracts from 8 day-old blood-fed A. gambiae

Salivary gland extracts were purified by ReadyPrep 2D Cleanup kit and 120 µg of proteins were

solubilized in 2D sample buffer, as described in the Methods section. Proteins were separated in

the first dimension using carrier ampholyte gradient gels between pH 4 and pH 8. Separation in

the second dimension was performed using 12.5% SDS acrylamide gel. The gel was stained

using SYPRO® Ruby.

Figure 4: Comparison of 8 day-old and 21 day-old salivary component functional

annotations.

A) 8 day-old salivary gland components; B) 21 day-old salivary gland components.

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Figure 5: Functional annotation of the 122 salivary components identified in 8 day-old and

21 day-old blood-fed Anopheles gambiae.

A) Contribution of various proteomic approaches to protein identification; B) Biological

processes in which they are involved.

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Table 1!: Proteins identified in salivary gland extracts of 8 day-old blood fed Anopheles gambiae

EnsemblIdentification (Ensemblrelease 43)

ProteinFamily/Description

PredictedMr/pI

Identification 1-DE-MS %coverage

2-DE-MSspot %number coveragea)

LC MS/MS Peptide sequence

Comments Subcellularlocalizationb)

Found inotherproteomic (P)ortranscriptomic (T) studies

Ensangp00000028522c) 30 kDa protein 26.9/3.8 2-DE-MS - 84, 184-188, 190,192-193,196-199,201-202

PSD EQELSDCIVDKRIKECFSSLDKELDDGLIEREQELSDCIVDKLMNPTIDLVSTIEKYSKECFSSLDKDVSAMVKKDDAEEDSEEGGEEGGDGASGGEGGEKESPR

GE rich salivarygland

secreted P [12], [9]

Ensangp00000018590 5 aminolevulinate synthaseerythroid specificmitochondrial precursor *

46.31/7.54 2-DE-MS - 110 25 - Metabolism ofamino acid

mitochondrialmatrix

this work

Ensangp00000015067 Ambiguous* 35.7/10.4 2-DE-MS - 186 16 - ? mitochondrial

this work

Ensangp00000015256 Ambiguous/candidate odorantreceptor*

44.85/7.01 LC MS/MS - - - AQRPVGITAGK Olfactoryreceptor(drosophila)

membranar this work

Ensangp00000022917 Ambiguous* 72.38/10.16 LC MS/MS - - - GRPILPLLKTVQSYK Tropomyosindomain

intracellular this work

Ensangp00000024702 Ambiguous* 30.31/9.58 LC MS/MS - - - IHDGVTHAAK ? ? this workEnsangp00000026134c) Ambiguous* 23.01/10 2-DE-MS - 169 PSD - ? ? this workEnsangp00000015382 Apyrase 61.79/8.6 1-DE-MS, LC

MS/MS,20% - - AAEEGDTCIAGIAR

LNVAQVAGLRGDITNEEAIGASPFSNTVDLLTLR

Anti-platelet secreted P [12]

Ensangp00000011707 Aspartate amino transferase* 44.71/6.78 2-DE-MS - 95 17 - Metabolism ofamino acid

cytoplasmic this work

Ensangp00000024137and/orEnsangp00000016868d)

ATP synthase subunit betamitochondrial precursor

22.69/4.9and/or19.72/5.27

2-DE-MS, LCMS/MS

- 66-67 (31-37) IINVIGEPIDERLVLEVAQHLGENTVR

Catalyzes ATPsynthesis

mitochondrial

P [12]

Ensangp00000018543 Chromosome associatedpolypeptide C XCAP Chomolog

156.83/5.34 LC MS/MS - - - LQTELIELKR Structuralmaintenance ofchromosomeABC transporterrelated domain

nuclear this work

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related domainEnsangp00000003518 CoA carboxylase

mitochondrial precursor*130.5/6.67 1-DE-MS 15% - - - Key enzyme in

the catabolicpathway of odd-chain aminoacids!:isoleucine,threonine,methionine andvaline

mitochondrial matrix

this work

Ensangp00000026391 Cofilin 16.93/7.28 2-DE-MS, LCMS/MS

- 170 42 LFLMSWCPDTAK Binds actin andassists intranslocation ofactin from thecytoplasm to thenucleusessential forcytokinesis,endocytosis andother cellprocesses thatrequire rapidturnover of actinfilaments

cytoplasmic T [9]

Ensangp00000022538 Creatine kinase 26.4/5.18 2-DE-MS - 90 30 - Phosphorylation cytoplasmic P [12]Ensangp00000025174and/orEnsangp00000018280d)

D7 precursor allergen AEDA2

35.57/5.7and/or32.7/5.1

1-DE-MS, 2-DE-MS, LCMS/MS

42% 114-119,

121-125,

149-151,

154, 169

(19-33) PSD

ALDPEEAWYVYERBVLIGLQLYEEKNYELSGSSQFKSADYAFLLRSANYGYLAMGKSDLEPEVRSVLASCTGTQAYDYYSCLLNSPVKDYELADSAEFRIYHGTVDSVAKNAFYFHELRNAMDCVFR

? secreted P [12], [9]

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Ensangp00000018340* D7 related-1 protein precursor 18.73/9.57 1-DE-MS, LCMS/MS

20% - - BLVESTSGEAFKKLPALSQYSSVVDKKVFDTVELVKCLVESTSGEAFK

Anti-inflammatoryScavenger ofbiogenic amines


Ensangp00000018371* D7 related-2 protein precursor 18.46/4.8 1-DE-MS, 2-DE-MS, LCMS/MS

28% 181-183 PSD ANTFYTCFLGTSSLAGFKESVLLELLQRHMQBVLEVVGFVDGNGEVKKANTFYTCFLGTSSLAGFKMQTSDPFDMNRNAVDYNELLKQYTPVSSDDMDK

Anti-inflammatory


Ensangp00000018330 D7 related-3 protein precursor 19.7/4.38 1-DE-MS, 2-DE-MS!, LCMS/MS

33% 180-181 PSD ANTFYTCFLGTSSAQAFKAGKLDMGTTFNAGQVSALMKLDMGTTFNAGQVSALMKYAVDYVELLR

Anti-inflammatory


Ensangp00000018328 D7 related-4 protein precursor 19.29/7.4 2-DE-MS, LCMS/MS

30% 171-176 (22-40) LYDPLNIIELDKCIGECVQVPTSERRYEIIEGPEMDKYTAEFVQIMKVFDLMELK

Anti-inflammatory

secreted P [12]

Ensangp00000027211 Disulfide isomerase precursor 54.31/5.47 2-DE-MS - 52 15 - Catalyzes therearrangement of-s-s- bonds inproteins

intracellular P [12]

Ensangp00000014287 Electron transfer flavoproteinalpha subunit mitochondrialpecursor*

34.14/8.62 2-DE-MS - 113 33 - Participates incatalyzing theinitial step of themitochondrialfatty acid beta-oxidation

mitochondrial

this work

Ensangp00000003806 Facilitated glucose transporter 16.83/8.48 LC MS/MS - - - HISQIVPLVAKGFSSKPLVP Sugar transporter membranar this workT [9]

Ensangp00000000937 probable Fatty acid bindingprotein

19.37/9.59 LC MS/MS - - - LGGGFDEETVDGR Fatty acidbinding protein


Ensangp00000016366 Precursor 45.95/9.43 2-DE-MS - 142 23 - Involved inenergy pathways


Ensangp00000011661 Glutathion S transferase (classtheta)

23.78/6.51 2-DE-MS - 155 33 - Key role incellulardetoxification

cytoplasmicand nuclear

This workP [50]

Ensangp00000024808 Glutathion S transferase 23.44/6.26 2-DE-MS - 156 23 - «! «! this workEnsangp00000010081 Glycogen phosphorylase 96.48/6.33 1-DE-MS 18% - - - Carbohydrate

metabolismcytoplasmic this work

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Ensangp00000009988 GSG3 20.01/4.34 2-DE-MS - 75, 76 PSD - ? secreted this workT [51]

Ensangp00000019455 GSG6 13.05/5.15 1-DE-MS, LCMS/MS

36% - - EPLPYMYACPGTEPCQSSDRETREPLPYMYACPGTEPCQSSDRSMHDVLCDRIDQAFLEQ

? secreted P [12], [9]

Ensangp00000021970 GSG7 16.29/8.46 LC MS/MS, - - - TLADETAQCMRTLADETAQCLRYGVQNQLR

? secreted P [12]

Ensangp00000005326 Guanine nucleotide releasingfactor

137.53/9.17 LC MS/MS - - - LIEKALIYK May play a rolein intracellularsignaling cascade

Membrane-associated

this work

Ensangp00000021028* putative gVAG proteinprecursor

28.9/8.96 1-DE-MS, LCMS/MS

43% - - DGQMDVYYFVBNYSFTNIMDRFPYAGQNIAITQFFGYRFVSSWWSEYLDARPEHVRGGPHVGCNPPSSSGGPTCQGKKYPSSYSGKPIGHFTQIASDRMPTLTWDPELASLADANARVGCSMWYWK

Allergen Belongs to theCAP family:proteaseinhibitors orproteolyticactivity, probablyinhibiting hostcoagulation orcomplementactivityDefence-relatedprotein


Ensangp00000017720 3 Hydroxyisobutyratedehydrogenase mitochondrial

34.31/9.27 LC MS/MS - - - VFADIVNASTGR Involved inamino acidcatabolismpathway

mitochondrial

this work

Ensangp00000016660 Isocitrate dehydrogenase 46.96/7.59 1-DE-MS 32% - - - Plays a key rolein cellulardefense againstoxidative stress-induced damage

mitochondrial

this work

Ensangp00000020184 Malate dehydrogenase 35.27/9.52 LC MS/MS - - - ANTFVGEAAGVDPQK Metabolicenzymes whichcatalyse the laststep in anaerobicglycolysis

mitochondrial

P [12]

Ensangp00000011006 Malate dehydrogenase 35.37/6.95 2-DE-MS 96 PSD DDLFNTNASIVR Participates inthe citric acidcycle


Ensangp00000017682 Maltase 67.21/5.87 1-DE-MS, 2-DE-MS, LCMS/MS

27% 6-8, 12 (17-43) AMPSGAIANWVLGNHDNSR Carbohydratedigestion

secreted P [12],

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DE-MS, LCMS/MS

DQPETYDMVHQWRELNVAAQLAAPRGITQTIDYLK

digestionConverts sucrosein nectar toglucose andfructose

Ensangp00000015067 Mitochondrial carrier 35.74/10.4 2-DE-MS - 186 16 -Ensangp00000011253 Nucleoside diphosphate kinase 19.01/8.46 1DE-MS, 32% - - GDLCVQVGR Maintenance of

cellular pool ofnucleosidetriphosphates

cytoplasmicand plasmamembrane

T [9]

Ensangp00000012716* putative 5’ Nucleotidaseprecursor

63.47/7.01 1-DE-MS, 2-DE-MS, LCMS/MS

20% 10, 13-33, 38-51, 54,57, 60,64, 65,77-82,85-87,130-134,140-141,144-145

(15-30)PSD

APFPLTLIHINDLHARDQIYYVVVPSYLADGKDGFAMKECIAGIARGLAPYLAELEKLGTQVIGTTEVFLDRESCRLSGADLWSAIDHSFTLDDEFRMKIPTVVANLEKNVNIIVVLSHCGLDGDKQLAEEAGDLIDVIVGAHSHSLLLNK


Ensangp00000028058 Peroxidase precursor 24.99/8.23 1-DE-MS, LCMS/MS

16% - - AFAGAININDHMFNPTVLERCFAIPVRPDDPVLSAGGIQCLDLVRLLPAEYGDGVYVPRSNITPELTILHVAFLRTTLVNMQFGQLVAHDMGLRWEDFVELR

Vasodilatator secreted P [12], [50]

Ensangp00000012460 Phosphoglycerate kinase 43.84/7.54 2-DE-MS - 109 27 - Glycolysis cytoplasmic this workEnsangp00000015800 Phosphoglycerate mutase 28.7/6.8 2-DE-MS, LC

MS/MS- 148 25 YGEEQVLIWR Involved in

energy pathwayscytoplasmic this work

Ensangp00000012492 Precursor 12.39/8.75 1-DE-MS 22% - - - EGF-like domain ? this workEnsangp00000013568 Precursor 41.83/5.4 2-DE-MS - 75 - - Aspartic protease

A1secreted this work

Ensangp00000016366 Precursor 45.95/9.43 2-DE-MS - 142 23 - Glucose-methanol-cholineoxidoreductaseInvolved inenergy pathways


Ensangp00000019046 Precursor 28.47/5.04 LC MS/MS - - - ANDRAMVK EGF-like domain ? this work

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Ensangp00000020734 Pterin 4 alpha carbinol aminedehydratase

21.20/10.23 LC MS/MS - - - LAQFLDQAAAVAK Transcriptionalactivator/pterindehydratase

? this work

Ensangp00000027538c) Retrovirus related polpolyprotein

9.51/11.28 2-DE-MS - 181, 183 PSD - ? nuclear this work

Ensangp00000021077 Ribonuclease 14.41/8.04 LC MS/MS ALAPYNQAIVADR Inhibits proteinsynthesis bycleavage ofmRNA

? this work

Ensangp00000027418* Salivary gland 1-like 3 44.51/6.04 1-DE-MS 30% - - - ? secreted P [12]

Ensangp00000018041 Toll precursor 16.69/4.51 2-DE-MS - 152 17 - Toll IAInvolved insignaltransductionpathways inresponse topathogens

plasmamembrane

P [50]

Ensangp00000018152 Triosephosphate isomerase 26.3/6.2 1-DE-MS, 2-DE-MS, LCMS/MS

30% - - AIFGETDELIAEKDWSNVVIAYEPVWAIGTGKSLLPETIGVAAQNCYKDLGLGWVILGHSER

Central enzymein the glycolyticpathwayPlays animportant role inseveral metabolicpathways

cytoplasmic this workP[52]

Ensangp00000012072 Unknown 29.21/4.43 2-DE-MS - 135 20 DSTLIMQLLR 14-3-3 protein.Family ofconservedregulatorymolecules thatbind a multitudeof functionallydiverse signalingproteins

cytoplasmic P [12]

Ensangp00000015472c) Unknown 15.64/10.38 1-DE-MS 20% - - - InterProZn-finger, C2H2typenucleic acid-binding protein

nuclear!? this workP [50], T [9]

Ensangp00000019887 Unknown 70.9/5.1 2-DE-MS - 9 18 - Heat shock 70regionMay be involvedin response tostress

cytoplasmicandorganelles

P [12]

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stressEnsangp00000028294 Unknown 15.18/4.57 LC MS/MS - - - GSTINLTBAVK Immunoglobulin-

like domainInvolved in celladhesion

Membrane!? this work

a) When several spots corresponded to the same protein, the percentage range of the sequence coverage is indicated in parenthesis. b) Subcellular localization is

inferred from sequence or structure similarity with orthologous proteins. c) Identification was performed using Ensembl database v35 of november 2005. d) Cases

where the same peptides match more than one genomic sequence. Shaded lines: proteins identified for the first time by a proteomic approach. * means that the

proteins were also identified in saliva. References underlined correspond to proteins found in human saliva.

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Table 2 : Proteins identified by LC MS/MS in salivary glands of 21 day-old blood-fed Anopheles gambiae

EnsemblIdentification (Ensembl release 43)


Predicted Mr/pI

Peptide sequence Comments Subcellular Localizationa)

Found in other proteomic (P) or transcriptomic (T) studies

Ensangp00000028522b), c), d)

30 kDa protein 26.90/3.8 EQELSDCIVDKRIKECFSSLDKELDDGLIEREQELSDCIVDKLMNPTIDLVSTIEKYSKECFSSLDKDVSAMVK

GE rich salivary gland secreted P [12] P [9]

Ensangp00000018525 Aconitate hydratase mitochondrial precursor

82.65/8.63 FDQNVYLPYEKISILGLNNFAPGK

Iron-sulphur proteins that function as electron carriersbiosynthesis of aminoacid

mitochondrial this work

Ensangp00000016546 Ambiguous 25.56/9.94 KGIGTHLMITLEVLAR GCN5-related N-acetyltransferasePutative role in transcription and DNA repair

? this work

Ensangp00000026066b)

Ambiguous 25.13/7.06 MSDKVVSSFLR ? ? this work

Ensangp00000027299 Ambiguous 339.53/6.98 EILYDDIERPILQTKLAGVFTPQEPLMNYVISCWVRQIVTFPDEERTAYLYDPQDVQLSVDGIVFRTFDETWATLAVRYPFGAGGEPFRLYFFASK

Subtilase serine protease domain? proteasome


Ensangp00000029258 Apolipoprotein D precursor

26.11/4.55 QSDVGRAVVAFPDESPLEAK Extracellular ligand-binding proteins displaying high specificity for small hydrophobic moleculesresponse to pathogens

secreted this work

Ensangp00000015382c

)

Apyrase 61.79/8.6 AAEEGDTCIAGIARLNVAQVAGLRGDITNEEAIGASPFSNTVDLLTLR


Ensangp00000026391b),c)

Cofilin 16.93/7.28 LFLMSWCPDTAK Binds actin and assists in translocation of actin from the cytoplasm to the nucleusessential for cytokinesis, endocytosis and other cell processes that require rapid

cytoplasmic T [9]

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turnover of actin filamentsEnsangp00000025174c

)D7 precursor allergen AED A2

35.57/5.7ou32.7/5.1


? secreted P [12], P [9]

Ensangp00000018340C

)D7 related-1 protein precursor

18.73/9.57 BLVESTSGEAFKKLPALSQYSSVVDKKVFDTVELVKCLVESTSGEAFK

Anti-inflammatoryScavenger of biogenic amines

secreted P [12], P [9]

Ensangp00000018371c

)

D7 related-2 protein precursor

18.46/4.8 ANTFYTCFLGTSSLAGFKESVLLELLQRHMQBVLEVVGFVDGNGEVKKANTFYTCFLGTSSLAGFKMQTSDPFDMNRNAVDYNELLKQYTPVSSDDMDK



Ensangp00000018330c


19.66/4.46 ANTFYTCFLGTSSAQAFKAGKLDMGTTFNAGQVSALMKLDMGTTFNAGQVSALMKYAVDYVELLR



Ensangp00000018328c

)


19.29/7.4 LYDPLNIIELDKCIGECVQVPTSERRYEIIEGPEMDKYTAEFVQIMKVFDLMELK


secreted P [12]

Ensangp00000018321 D7 related-5 protein precursor

18.79/5.82 SGSFFSCMLR ? secreted P [12]

Ensangp00000003578 GSG5 precursor 38.2/6.42 TYFQNEFVEYR ? secreted T [51]

Ensangp00000019455c

)GSG6 13.05/5.15 EPLPYMYACPGTEPCQSSDR

ETREPLPYMYACPGTEPCQSSDRSMHDVLCDRIDQAFLEQ


Ensangp00000021970c

)GSG7 16.29/8.46 TLADETAQCMR

TLADETAQCLR? secreted P [12]

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YGVQNQLREnsangp00000021028c

)

putative gVAG protein precursor

28.9/8.96 DGQMDVYYFVBNYSFTNIMDRFPYAGQNIAITQFFGYRFVSSWWSEYLDARPEHVRGGPHVGCNPPSSSGGPTCQGKKYPSSYSGKPIGHFTQIASDRMPTLTWDPELASLADANARVGCSMWYWK

AllergenBelongs to the CAP family: protease inhibitors or proteolytic activity, probably inhibiting host coagulation or complement activityDefence-related protein


Ensangp00000009655 Homolog 118.45/6.27 DGKELDLVCMQK C2 domain (cellular proteins involved in signal transduction or membrane trafficking)Cytochrome c heme-binding site (electron-transfer proteins)

? this work

Ensangp00000018375 Hypothetical 10 kD protein

10/6.22 LSLQLEEFAVCKAISDLQQGLFDLNHCTK

? secreted this work

Ensangp00000018379 Hypothetical 10.2 kD protein

10.13/4.52 LQQMVEDFTACR ? secreted P [12]

Ensangp00000004315 Hypothetical 8.8 kDa 8.82/4.05 DKPDIDPVDFLVDVIK ? secreted P [12]

Ensangp00000020384 Low density lipoprotein receptor

17.3/5.04 CISRAGICDGK Lipid metabolism membranar P [50]

Ensangp00000022875 Lysozyme precursor 15.33/8.56 NGSTDYGIFQINNKYWBDSGYGSNDCKNLLNDDITDDIKKLPNVSSCF

Immunity relatedAntibacterial enzyme


Ensangp00000020184c

)Malate dehydrogenase

35.27/9.52 ANTFVGEAAGVDPQK Metabolic enzymes which catalyse the last step in anaerobic glycolysis

mitochondrial P [12]

Ensangp00000017682c

)Maltase 67.21/5.87 AMPSGAIANWVLGNHDNSR

DQPETYDMVHQWRELNVAAQLAAPRGITQTIDYLK

Sugar digestionConverts sucrose in nectar to glucose and fructose

secreted P [12]

Ensangp00000004215 Mitogen activated kinase kinase kinasekinase

159.64/10.04 NIATYYGAFIK Protein kinaseATP binding


Ensangp00000003978 N acylneuraminate cytidyltransferase

21.1/5.67 HLTLARILLGME Forms CMP-NeuAc, the nucleotide sugar donor used by sialyltransferases (modification may be important in pathogenesis)


Ensangp00000021120 NADPH dependent carbonyl reductase

27.05/7.73 MDFTGKVVLITGASSGIGASTAK Sugar metabolism cytoplasmic this work

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Ensangp00000012716c

)Putative 5’ nucleotidase precursor

63.47/7.01 APFPLTLIHINDLHARDQIYYVVVPSYLADGKDGFAMKECIAGIARGLAPYLAELEKLGTQVIGTTEVFLDRESCRLSGADLWSAIDHSFTLDDEFRMKIPTVVANLEKNVNIIVVLSHCGLDGDKQLAEEAGDLIDVIVGAHSHSLLLNK


Ensangp00000020778 Peptidyl prolyl cis trans isomerase

18.29/8.97 FFDMTVDNQPLGRIVIELRPDVVPKHVVFGSVVEGMDVVR

Accelerates protein folding cytopasmic this work

Ensangp00000028058c

)Peroxidase precursor 24.99/8.23 AFAGAININDHMFNPTVLER

CFAIPVRPDDPVLSAGGIQCLDLVRLLPAEYGDGVYVPRSNITPELTILHVAFLRTTLVNMQFGQLVAHDMGLRWEDFVELR

Vasodilatator secreted P [12], P [50]

Ensangp00000015800c

)Phosphoglycerate mutase

28.7/6.8 YGEEQVLIWR Involved in energy pathways cytoplasmic this work Table 1

Ensangp00000029324 Precursor 25.94/4.8 TLTFVLKPTK Alpha 2 macroglobulin domain intracellular this work

Ensangp00000021077c

)Ribonuclease 14.41/8.04 ALAPYNQAIVADR Inhibits protein synthesis by

cleavage of mRNA? this work Table 1

Ensangp00000019607 Ryanodine receptor 1 577.53/5.18 YFDMFLKLK Ca2+ release channels involved in secretory pathways ?

membranar this work

Ensangp00000008103 Stromal interaction molecule precursor

54.49/6.36 DVEGLLKAEVALK ? membranar this work

Ensangp00000028309 Trans enoyl COA isomerase mitochondrial precursor

30.18/7.13 ALEQAVAFLNR Fatty acid metabolism mitochondrial this work

Ensangp00000018152c

)Triosephosphate isomerase

22.52/5.09 AIFGETDELIAEKDWSNVVIAYEPVWAIGTGKSLLPETIGVAAQNCYKDLGLGWVILGHSER

Central enzyme in the glycolytic pathwayPlays an important role in several metabolic pathways

cytoplasmic this work Table 1

Ensangp00000000334b Unknown 39.57/7.29 SPILLLDDIFDK ATP/GTP-binding site motif A intracellular this work

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) (P-loop)Ensangp00000011593 Wilm’s tumor 1

associating WT1 associated splicing regulator female lethal 2-D homolog

32.55/4.78 FTPDSNTGKR Potential role in transcriptional regulationInvolves in alternative splicing regulation

nuclear this work

a) Subcellular localization is inferred from sequence or structure similarity with orthologous proteins. b) Identification was performed using

Ensembl database v35 of november 2005. c) proteins identified from salivary gland extracts of young blood-fed females. Shaded lines: Proteins

identified for the first time by a proteomic approach.

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Table 3 : List of proteins identified in salivary gland extract of 21day-old blood-fed Anopheles gambiae using iTRAQ

EnsemblIdentification(Ensembl release 43)


PredictedMr/pI

Ratio 117/114a) Peptide sequence Comments SubcellularLocalizationb)

Found inotherproteomic (P) ortranscriptomic (T)studies

Ensangp00000028522c),d)

30 kDa protein 26.90/3.8 1.3 ± 0.5 EQELSDCIVDKRIKECFSSLDKELDDGLIEREQELSDCIVDKEGEEGAGSDDAVSGADDETEESKDDAEEDSEEGGEEGGDGASGGEGGEKESPRLMNPTIDLVSTIEKYSKECFSSLDKDVSAMVK

GE rich salivary gland secreted P [12], P [9]

Ensangp00000022344 30 kDa protein 18.7/3.7 - EGEEGAGSDDAVSGADDETEESKDDAEEDSEEGGEEGGDGASGGEGGEKESPR

GE rich salivary gland

Ensangp00000018525 Aconitate hydratasemitochondrialprecursor

82.65/8.63

- FDQNVYLPYEKISILGLNNFAPGK

Iron-sulphur proteins thatfunction as electron carriersbiosynthesis of amino acid

mitochondrial this work Table 2

Ensangp00000019171 Acyl-coA-bindingprotein

9.85/9.45 - RPSDAELLELYALFK May act as an intra-cellularcarrier of acyl-CoA esters


Ensangp00000031876 Acyl-coA-bindingprotein

9.65/7.35 - NLNATPADADLLEIYGLFJ « « this work

Ensangp00000017843 Alanine aminotransferase 2

52.54/7.79

- ANIGDCHAMGQPPITFIR Metabolism of amino acid cytoplasmic this work

Ensangp00000026558c

)Ambiguous* 124.54/8.

43- STTAALLISVLVR ? ? this work

Ensangp00000027299 Ambiguous 339.53/6.98

- EILYDDIERPILQTKLAGVFTPQEPLMNYVISCWVRQIVTFPDEERTAYLYDPQDVQLSVDGIVFRTFDETWATLAVRYPFGAGGEPFRLYFFASK

Subtilase serine protease? proteasome

cytoplasmic this work Table 2

Ensangp00000015145and/orEnsangp00000012963e

)

Annexin 35.57/4.31and/or27.25/4.11

- LLTMIIVGAR Inhibit PLA2 activity,involved in exocytosiscalcium-dependentphospholipid-binding proteins


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Ensangp00000015382 Apyrase 61.79/8.6 0.71±0.11 AAEEGDTCIAGIARLNVAQVAGLRGDITNEEAIGASPFSNTVDLLTLR


Ensangp00000024604 ATP synthasesubunit alphamitochondrialprecursor

59.45/9.52

- GAEISAILEER Catalyzes ATP synthesis mitochondrial P [12]

Ensangp00000024137and/orEnsangp00000016868e)

ATP synthasesubunit betamitochondrialprecursor

22.69/4.9and/or19.72/5.27

* IINVIGEPIDERLVLEVAQHLGENTVR

Catalyzes ATP synthesis mitochondrial P [12]

Ensangp00000012700 Calmodulin 17.25/3.99

- EAFSLFDKDGDGTITTKVFDKDGNGFISAAELRGQNPTEAELQDMINEVDADGNGTTTKELGTIDFPEFLTMADGNGTIDFPGTITTKELGTVEEVDEMIREADIDFPEFLTMMARADQLTEEQIAEFKDMINEVDADGNGTQVNYEARILHLIKFSLFDKDGDGTITTDADGNGTIDFPEFLAFSLFDKDGDGTITTK

Calcium binding protein intracellular T [53]

Ensangp00000026391 Cofilin 16.93/7.28

- LFLMSWCPDTAK Binds actin and assists intranslocation of actin from thecytoplasm to the nucleusessential for cytokinesis,endocytosis and other cellprocesses that require rapidturnover of actin filaments

cytoplasmic This work Tables 1 and2T [9]

Ensangp00000022538 Creatine kinase 26.4/5.18 * AVQQQLIDDHFLFKTFLVWCNEEDHLR

Phosphorylation cytoplasmic P [12]

Ensangp00000020091 Cytochrome c 11.78/10.17

- GDLIAYLK Electron tranporter Mitochondrialmembrane

this work

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Ensangp00000025174and/orEnsangp00000018280e)

D7 precursorallergen AED A2

35.57/5.7and/or32.7/5.1

0.77±0.05 ALDPEEAWYVYERBVLIGLQLYEEKNYELSGSSQFKSADYAFLLRSANYGYLAMGKSDLEPEVRSVLASCTGTQAYDYYSCLLNSPVKDYELADSAEFRIYHGTVDSVAKNAFYFHELRNAMDCVFR


Ensangp00000018340 D7 related-1 proteinprecursor

18.73/9.57

0.67±0.07 BLVESTSGEAFKKLPALSQYSSVVDKKVFDTVELVKCLVESTSGEAFK




18.46/4.8 0.92±0.08 ANTFYTCFLGTSSLAGFKESVLLELLQRHMQBVLEVVGFVDGNGEVKKANTFYTCFLGTSSLAGFKMQTSDPFDMNRNAVDYNELLKQYTPVSSDDMDK



Ensangp00000018330and/orEnsangp00000025580d

), e)

D7 related-3 proteinprecursor

19.66/4.46and/or18.6/4.5

0.95±0.15 ANTFYTCFLGTSSAQAFKAGKLDMGTTFNAGQVSALMKLDMGTTFNAGQVSALMKYAVDYVELLR



Ensangp00000018328d

)

D7 related-4 proteinprecursor

19.29/7.4 0.9±0.05 LYDPLNIIELDKCIGECVQVPTSERRYEIIEGPEMDKYTAEFVQIMKVFDLMELK


secreted P [12]


18.79/5.82

- SGSFFSCMLR ? secreted P [12]

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Ensangp00000018385c

)

Disulfide isomeraseprecursor

54.31/5.47

- QGETDAVFLFR Catalyzes the rearrangement of-s-s- bonds in proteins


Ensangp00000026077 Disulfide isomeraseprecursor

55.41/4.61

* ELETVEAAEEFLKILEFFGMKILEFVQSFLDGK

Catalyzes the rearrangement of-s-s- bonds in proteins

endoplasmicreticulumlumen

P [12]

Ensangp00000002028 DNA2 helicase 117.99/8.45

- EKLIIIGDR ATP binding ? this work

Ensangp00000014287d

)Electron transferflavoprotein subunitalpha mitochondrialpecursor

34.14/8.62

* FTHIVAGATAFGK Participates in catalyzing theinitial step of the mitochondrialfatty acid beta-oxidation


Ensangp00000018531 Enolase 46.62/6.9 * AAVPSGASTGVHEALELREALNLIQDAIAKGNPTVEVDLVTDLGLFR

Glycolytic enzyme cytoplasmic* P [12]

Ensangp00000010297 Enzyme 79.47/9.67

- LTSIPTALDLALTGK Includes enoyl coA hydrataseinvolved in fatty-acidmetabolism


Ensangp00000024159 Fructose biphosphatealdolase

39.18/7.72

* KPTAQEIALATVTALRIVPIVEPEILPDGDHDLER

Glycolytic enzyme ? P [12], P [50]

Ensangp00000020828 Fumarasemitochondrialprecursor

50.22/7.55

- IADAIALAADDVISGK Amino acid metabolism ? mitochondrial this work

Ensangp00000017396 Fumaryl acetoacetase

45.64/6 - GTKQVSLAGGETR Last enzyme of the tyrosinecatabolic pathway


Ensangp00000029040 Glutathion Stransferase

19.16/7.5 * LYFDMGTLYQR « « this work

Ensangp00000010360 Glyceraldehydephosphatedehydrogenase

35.46/8.55

- AGAEYVVESTGVFTTTEKWRDGKLTGMGCLVVNASVVAIIIPAATGHATTATQKTAFRVPTPNVSLSKPATYDQIGAAKAVGKVIP

Plays an important role inglycolysis and gluconeogenesis

cytoplasmic P [12], P [50]

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Ensangp00000003578 GSG5 38.2/6.42 - TYFQNEFVEYR ? secreted This work Table 2T [51]

Ensangp00000019455 GSG6 13.05/5.15

0.65±0.05 EPLPYMYACPGTEPCQSSDRETREPLPYMYACPGTEPCQSSDRSMHDVLCDRIDQAFLEQ


Ensangp00000021970 GSG7 16.29/8.46

- TLADETAQCMRTLADETAQCLRYGVQNQLR

? secreted P [12]

Ensangp00000021028 putative gVAGprotein precursor

28.9/8.96 1.97± 0.6 DGQMDVYYFVBNYSFTNIMDRFPYAGQNIAITQFFGYRFVSSWWSEYLDARPEHVRGGPHVGCNPPSSSGGPTCQGKKYPSSYSGKPIGHFTQIASDRMPTLTWDPELASLADANARVGCSMWYWK

AllergenBelongs to the CAP family:protease inhibitors orproteolytic activity. probablyinhibiting host coagulation orcomplement activity


Ensangp00000014839 60 kDa heat shockproteinmitochondrialprecursor

60.77/5.28

- VEFQDALVLFSEK Protein refolding mitochondrial this work

Ensangp00000003808 HistoneacetyltransferaseGCN5

85.65/8.9 - SIPIESIPGLR Control of amino acid synthesis nuclear this work

Ensangp00000009655 Homolog 118.45/6.27

- DGKELDLVCMQK C2 domain (cellular proteinsinvolved in signal transductionor membrane trafficking)Cytochrome c heme-bindingsite (electron-transfer proteins)

? this work Table 2

Ensangp00000004315 Hypothetical 8.8 kDaprotein

8.82/4.05 * DKPDIDPVDFLVDVIK ? secreted P [12]

Ensangp00000018375 Hypothetical 10 kDaprotein

10/6.22 - LSLQLEEFAVCKAISDLQQGLFDLNHCTK

? secreted this work Table 2

Ensangp00000018379 Hypothetical 10.2kDa protein

10.13/4.52

- LQQMVEDFTACR ? secreted P [12]

Ensangp00000013285 3 Ketoacyl coAthiolase

41.67/8.47

* AALDAAGLKPDQVDSVNIGQVLVLSSTDGAFLPRLACAGELGLDINKLNLNGAQDILVGAAHTAGTASGIASGSRITG

Involved in biosyntheticpathways such as poly beta-hydroxybutyrate synthesis orsteroid biogenesis


Ensangp00000010689 Kinase 74.81/9.33

- SLDLLDSMLVLDPPGSEDLSGEEDIGSPLLPSNRDTIQNLTPSGREIKILRQ

Protein phosphorylation cytoplasmic this work

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AGINMMGGAGAPAG

Ensangp00000020132 Low densitylipoprotein receptor

179.24/6.29

- DGTERVLIVSQNLGSQRVELITKIVTAEIQAPDGSPDDAPADHVCACPQGLMLLKGRTN

Lipid metabolism membranar this work

Ensangp00000022875 Lysozyme precursor 15.33/8.56

* NGSTDYGIFQINNKYWBDSGYGSNDCKNLLNDDITDDIKKLPNVSSCF



Ensangp00000020184 Malatedehydrogenase

35.27/9.52

- ANTFVGEAAGVDPQK Metabolic enzymes whichcatalyse the last step inanaerobic glycolysis


Ensangp00000017682 Maltase 67.21/5.87

1.3±0.2 AMPSGAIANWVLGNHDNSRDQPETYDMVHQWRELNVAAQLAAPRGITQTIDYLK

Sugar digestionConverts sucrose in nectar toglucose and fructose

secreted P [12]

Ensangp00000003748 Myosin 121.89/10.23

- Contractile protein cytoplasmic this workP [52]

Ensangp00000026137 Nucleolar RNAassociated protein

117.56/7.05

- LSSETIDELEK Appears to be associated withribosome biogenesis


Ensangp00000011253 Nucleosidediphosphate kinase

19.01/8.46

- GDLCVQVGR Maintenance of cellular pool ofnucleoside triphosphates

cytoplasmicand plasmamembrane

this work Table 1T [9]

Ensangp00000012716 Putative 5’nucleotidaseprecursor

63.47/7.01

0.92±0.24 APFPLTLIHINDLHARDQIYYVVVPSYLADGKDGFAMKECIAGIARGLAPYLAELEKLGTQVIGTTEVFLDRESCRLSGADLWSAIDHSFTLDDEFRMKIPTVVANLEKNVNIIVVLSHCGLDGDKQLAEEAGDLIDVIVGAHSHSLLLNK


Ensangp00000020778 Peptidyl prolyl cistrans isomerase

18.29/8.97

- FFDMTVDNQPLGRIVIELRPDVVPKHVVFGSVVEGMDVVR

Accelerates protein folding cytopasmic this work

Ensangp00000028058 Peroxidase precursor 24.99/8.23

0.95±0.15 AFAGAININDHMFNPTVLERCFAIPVRPDDPVLSAGGIQCLDLVRLLPAEYGDGVYVPR

Vasodilatator secreted P [12], P[50]

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SNITPELTILHVAFLRTTLVNMQFGQLVAHDMGLRWEDFVELR

Ensangp00000020634 Peroxysomaltargeting signal 2receeptor

36.41/6.22

- VSGSGDGSVQLWNTFTTNRTNLASSVQLWNTNLASNSQFYGLAGGGT

Family of potein implicated ina variety of functions rangingfrom signal transduction andtranscription regulation to cellcycle control and apoptosis

? this work

Ensangp00000024749 Pheromone/generalodorant bindingprotein OBP56

27.15/5.52

- SASEVQDDKCK ? ? this work

Ensangp00000013993 Phosphatidylethanolamine-binding protein

24.17/6.67

- YVFLVYK Proteinase inhibitor ? this workP [52]

Ensangp00000020531 Precursor 200.9/4.5 - ERTGEIMLLQRAGTIVGNVSALDEDVGPNGTRDARLDRDTNPESYAIGTIFVNSTLNYNYAAVIVERQLDYEEVSGVLDRFTVEMQERLANANLELS

Cadherin membranar this work

Ensangp00000031578 Precursor 58.96/9.68

- DMPNITLLNLDGNQLSRNLLQNLDLALFVAMPQLLNLNASSPVANNLTSAPIAPVTGRPNITLLNVSAPIGLNKITTFNIT

Leucine rich repeatPutatively involved in protein-protein interaction

? this work

Ensangp00000021077 Ribonuclease 14.41/8.04

- ALAPYNQAIVADR Inhibits protein synthesis bycleavage of mRNA

? this work

Ensangp00000006850 DNA directed RNApolymerase

68.25/8.18

- LSYISALGMMTR Transcription nuclear this work

Ensangp00000017327 Putative salivaryprotein GSG1b

46.6/7.37 - DYESYLGAMFAADAFHVVYEADGK

? secreted P [12]

Ensangp00000032098d

)Salivary D3 protein ? - AAAGPAPDPSSQFCQQLLDDAQ

RSaglin secreted P [12]

Ensangp00000020530 Serine proteaseprecursor

25.2/4.57 - NGQNDIALLQLDRKVITSAQCTTDEGNGIPSVVRLGGTK

Involved in immunity or incoagulation cascade

secreted this work

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SVLFAVLLIWDSVVALLQLDRKIIINTTDEGNGIPSVVR

Ensangp00000016680 Serpin 9 46.36/7.01

- LAAETDILHEVVNEGISR Serine protease inhibitorInvolved in immunity

secreted T [11]

Ensangp00000009988 SG3 4.3/20.013

- ATGPLFLPHFGQGPRRGQQLIFLAASVERNPAATIAVASAATASPTTAEAQQQRQQVQR

Mucin secreted T [51]

Ensangp00000009009 Fact complex subunitfacilitates chromatintrancription

71.65/6.28

- RPLSAYMLWLNSAR Recombination signal sequencerecognition T160

nuclear this work

Ensangp00000016164 Superoxydedismutase

15.67/5.45

- SLVVHADPDDLGVGGHELSK Metalloprotein that preventsdamage by oxygen-mediatedfree radicals


Ensangp00000021085 Translationallycontrolled tumorprotein TCTP

19.54/4.42

- LVDDVMYEVYGK Histamine-releasing factor ? T [9], T [11]

Ensangp00000017522 Trio protein 43.78/6.4 - SMYDLIGQLVQSSK ? secreted ? P [12]

Ensangp00000025045 Trypsin precusror 28.65/6.49

- QIGIVSWGDTQCVGTRGGSSTLNETDLTVRRLALTAGHNGNFVPNLPAPARATGRIV

Proteolytic enzyme secreted this work

Ensangp00000008105 E3 Ubiquitin ligase 201.24/6.46

- GLAMADLDRLEKQQLCIKPNPDNSEHRNHKGTYHSVNTQASQQQQAPLLRDGSRVMMMG

Involved in protein degradationpathway


Ensangp00000012822 Unknown 74.9/7.88 * DVQASHISRLGTSSIVSYTPTLRNGTPQASNSIYCTLRNGTNVSMCPDTIDSD

Immunoglobulin-like ? this work

Ensangp00000012893 Unknown 72.74/4.9 - ELEDIVQPIIAK Hsp70 and tropomyosin ER ? this work

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2 domainsEnsangp00000016832 Unknown 19.42/4.8

8- QQAAAAAETTSQAAGTLMDHA

KAnti-freeze protein ? this work

Ensangp00000017135 Unknown 85.43/8.64

- IKCGLLLEGVR ? ? this work

Ensangp00000019537 Unknown 90.81/7.41

* KLMSDYYSSVVASTNEMQSLFLPSSQREHMAHSQTGSTT

? ? this work

Ensangp00000028177 Unknown 36.81/10.03

- LGIGSSSINGSGAVVRK Basic helix-loop-helixdimerisation region

this work

Ensangp00000029447 Unknown 20.35/6.24

- EQQQLALDVR ? secreted this work

a) Ratios indicated in bold correspond to a significant increase or decrease of protein expression in the presence of Plasmodium. b) Subcellular

localization is inferred from sequence or structure similarity with orthologous proteins. c) Identification was performed using Ensembl database

v35 of november 2005. d) The part of the sequence in bold is that described in Ensembl 43. e) Cases where the same peptides match more than

one genomic sequence. * means that the protein was quantified one time. Shaded lines : Proteins newly identified by iTRAQ. References

underlined correspond to proteins found in human saliva.

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Supplementary Table 1 : List of proteins identified by 1-DE-MS according to their slice

number

Slice number Ensembl identification Protein family /description

1 No signal 2 Ensangp00000003518 CoA carboxylase 3 Ensangp00000010081

and Ensangp00000012716

Glycogen phosphorylase and putative 5’ nucleotidase precursor

4 Ensangp00000012716 and Ensangp00000017682

putative 5’ nucleotidase precursor and Maltase

5 Ensangp00000012716 putative 5’ nucleotidase precursor 6 Ensangp00000017682 Maltase 7 Ensangp00000012716 putative 5’ nucleotidase precursor 8 Ensangp00000012716


putative 5’ nucleotidase precursor and Apyrase

9 Ensangp00000016660 Isocitrate dehydrogenase 10 NI 11-12 Ensangp00000025174 D7 precursor allergen AED A2 13 Ensangp00000027418 Salivary gland 1-like 3 14-15 Ensangp00000018280/25174 D7 precursor allergen AED A2 16 Ensangp00000018280/25174


D7 precursor allergen AED A2 and Putative gVAG protein precursor

17 Ensangp00000018280 D7 precursor allergen AED A2 18 NI 19 Ensangp00000018152 Triosephosphate isomerase 20-22 Ensangp00000011253

and Ensangp00000021028 and Ensangp00000018328

Nucleoside diphosphate kinase and putative gVAG protein precursor and D7 related-4 protein precursor

23 Ensangp00000018328 and Ensangp00000018330 And Ensangp00000018340

D7 related-4 protein precursor and D7 related-3 protein precursor and D7 related-1 protein precursor

24-25 Ensangp00000018371 and Ensangp00000018330

D7 related-2 protein precursor and D7-related-3 protein precursor

26-27 Ensangp00000018371 and Ensangp00000018330 and Ensangp00000019455

D7 related-2 protein precursor and D7 related-3 protein precursor and GSG6

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D7 related-2 protein precursor and D7 related-3 protein precursor


D7 related-2 protein precursor and precursor

NI : non-identified protein

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Supplementary Table 2 : List of proteins identified by 2-DE-MS according to their spot

number

Spot number Ensembl identification Protein family /description1-5 NI6-8 Ensangp00000017682 Maltase 9 Ensangp00000019887 unknown10 Ensangp00000012716 putative 5’ nucleotidase precursor11 NI12 Ensangp00000017682 Maltase 13-33 Ensangp00000012716 putative 5’ nucleotidase precursor34-37 NI38-51 Ensangp00000012716 putative 5’ nucleotidase precursor52 Ensangp00000027211 Disulfide isomerase precursor53 NI54 Ensangp00000012716 putative 5’ nucleotidase precursor55-56 NI57 Ensangp00000012716 putative 5’ nucleotidase precursor58-59 NI60 Ensangp00000012716 putative 5’ nucleotidase precursor61-63 NI64-65 Ensangp00000012716 putative 5’ nucleotidase precursor66-67 Ensangp00000024137

and/orEnsangp00000016868

ATP synthase subunit beta mitochondrial precursor

68-74 NI75 Ensangp00000013568

andEnsangp00000009988

PrecursorandGSG3

76 Ensangp00000009988 GSG377-82 Ensangp00000012716 putative 5’ nucleotidase precursor83 NI84 Ensangp00000028522 30 kDa85-87 Ensangp00000012716 putative 5’ nucleotidase precursor88-89 NI90 Ensangp00000022538 Creatine kinase91-94 NI95 Ensangp00000011707 Aspartate amino transferase96 Ensangp00000011006 Malate dehydrogenase97-108 NI109 Ensangp00000012460 Phosphoglycerate kinase110 Ensangp00000018590 5 aminolevulinate synthase111-112 NI113 Ensangp00000014287


Electron transfer flavoprotein alpha subunitandD7 precursor allergen AED A2

114-119 Ensangp00000025174 D7 precursor allergen AED A2120 NI121-125 Ensangp00000025174 D7 precursor allergen AED A2

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126-129 NI130-134 Ensangp00000012716 putative 5’ nucleotidase precursor135 Ensangp00000012702 Unknown136-139 NI140-141 Ensangp00000012716 putative 5’ nucleotidase precursor142 Ensangp00000016366 Glucose dehydrogenase precursor143 NI144-145 Ensangp00000012716 putative 5’ nucleotidase precursor146 NI147 NI148 Ensangp00000015800 Phosphoglycerate mutase149-151 Ensangp00000025174 D7 precursor allergen AED A2152 Ensangp00000018152


Triose phosphate isomeraseandToll precursor

153 NI154 Ensangp00000025174 D7 precursor allergen AED A2155 Ensangp00000011661 Glutathion S transferase156 Ensangp00000024808 Glutathion S transferase157-168 NI169 Ensangp00000026134


AmbiguousandD7 precursor allergen AED A2

170 Ensangp00000026391 cofilin171-176 Ensangp00000018328 D7 related-4 protein precursor177-179 NI180 Ensangp00000018330 D7 related-3 protein precursor181 Ensangp00000018371

andEnsangp00000027538andEnsangp00000018330

D7-related-2 protein precursorandRetrovirus related pol polyproteinandD7 related-3 protein precursor

182-183 Ensangp00000018371andEnsangp00000027538

D7 related-2 protein precursorandRetrovirus related pol polyprotein

184-185 Ensangp00000028522 30 kDa186 Ensangp00000015067


Mitochondrial carrierand30 kDa

187-202 Ensangp00000028522 30 kDa203-205 NI

NI : non-identified protein

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Supplementary Table 3: Proteins identified in salivary gland extracts of A. gambiae blood-fed females

EnsemblIdentification (Ensembl release 43)


Predicted Mr/pI

Identification 1DE-MS% coverage

2-DE-MSspot %number coveragea)

LC MS/MS Peptide sequence

Comments Subcellular Localizationb)

Found in other proteomic (P) or transcriptomic (T) studies

Ensangp00000028522c

)

Ensangp00000022344d)

30 kDa protein 26.90/3.818.7/3.7

2-DE-MSLC MS/MS iTRAQ

- 84, 184-188, 190, 192,-193, 196-199, 201-202

PSD EQELSDCIVDKRIKECFSSLDKELDDGLIEREQELSDCIVDKLMNPTIDLVSTIEKYSKECFSSLDKDVSAMVKEGEEGAGSDDAVSGADDETEESKDDAEEDSEEGGEEGGDGASGGEGGEKESPR

GE rich salivary gland


Ensangp00000018525e

)Aconitate hydratase mitochondrial precursor

82.65/8.63

LC MS/MSiTRAQ

- - - FDQNVYLPYEKISILGLNNFAPGK

Iron-sulphur proteins that function as electron carriersbiosynthesis of amino acid


Ensangp00000019171e

)Acyl-coA -binding protein

9.85/9.45 iTRAQ - - - RPSDAELLELYALFK May act as an intra-cellular carrier of acyl-CoA esters


Ensangp00000031876e

)Acyl-coA -binding protein

9.65/7.35 iTRAQ - - - NLNATPADADLLEIYGLFJ « « this work

Ensangp00000017843e

)Alanine aminotransferase 2

52.54/7.79

iTRAQ - - - ANIGDCHAMGQPPITFIR Metabolism of amino acid


Ensangp00000016546e

)Ambiguous 25.56/9.9

4LC MS/MS - - - KGIGTHLMITLEVLAR GCN5-

related N-acetyltransferase

? this work

Ensangp00000022917d

)Ambiguous 72.38/10.

16LC MS/MS - - - GRPILPLLKTVQSYK Tropomyosin

domainintracellular this work

Ensangp00000024702d


8LC MS/MS - - - IHDGVTHAAK ? ? this work

Ensangp00000026066c


6LC MS/MS - - - MSDKVVSSFLR ? ? this work

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Ensangp00000026134c

),d)Ambiguous 23.01/10 2-DE-MS - 169 PSD - ? ? this work

Ensangp00000026558c


43LC MS/MS - - - STTAALLISVLVR ? ? this work

Ensangp00000027299e


98LC MS/MS, ITRAQ

- - - EILYDDIERPILQTKLAGVFTPQEPLMNYVISCWVRQIVTFPDEERTAYLYDPQDVQLSVDGIVFRTFDETWATLAVRYPFGAGGEPFRLYFFASK

Subtilase serine protease


Ensangp00000018590d

)5 Aminolevulinatesynthase erythroid specific mitochondrial precursor

46.31/7.54

2-DE-MS - 110 25 - Metabolism of amino acid

mitochondrialmatrix

this work

Ensangp00000015145and/orEnsangp00000012963e),f)

Annexin 35.57/4.31and/or27.25/4.11

iTRAQ - - - LLTMIIVGAR Inhibit PLA2 activity,involved in exocytosiscalcium-dependent phospholipid-binding proteins


Ensangp00000029258e

)Apolipoprotein D precursor

26.11/4.55

LC MS/MS - - - QSDVGRAVVAFPDESPLEAK Extracellular ligand-binding proteins displaying high specificity for small hydrophobic molecules

secreted this work

Ensangp00000015382d

),e)Apyrase 61.79/8.6 1-DE-MS

LC MS/MS iTRAQ

20% - - AAEEGDTCIAGIARLNVAQVAGLRGDITNEEAIGASPFSNTVDLLTLR


Ensangp00000011707d

)Aspartate aminotransferase

44.71/6.78

2-DE-MS - 95 17 - Metabolism of amino acid


Ensangp00000024604e

)ATP synthase subunit alpha

59.45/9.52

iTRAQ - - - GAEISAILEER Catalyzes ATP


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mitochondrial precursor

synthesis

Ensangp00000024137and/orEnsangp00000016868d),e),f)

ATP synthase subunit beta mitochondrial precursor

22.69/4.9and/or19.72/5.27

LC MS/MS iTRAQ

- 66-67 (31-37) IINVIGEPIDERLVLEVAQHLGENTVR

Catalyzes ATP synthesis


Ensangp00000012700e

)Calmodulin 17.25/3.9

9iTRAQ - - - EAFSLFDKDGDGTITTK

VFDKDGNGFISAAELRGQNPTEAELQDMINEVDADGNGTTTKELGTIDFPEFLTMADGNGTIDFPGTITTKELGTVEEVDEMIREADIDFPEFLTMMARADQLTEEQIAEFKDMINEVDADGNGTQVNYEARILHLIKFSLFDKDGDGTITTDADGNGTIDFPEFLAFSLFDKDGDGTITTK

Calcium binding protein


Ensangp00000018543d

)Chromosome associated polypeptide C XCAP C homolog

156.83/5.34

LC MS/MS - - - LQTELIELKR Structural maintenance of chromosomeABC transporter related domain

nuclear this work

Ensangp00000003518d

)CoA carboxylase mitochondrial precursor

130.5/6.67

1-DE-MS 15% - - - Key enzyme in the catabolic pathway of odd-chain fatty acids : isoleucine, threonine, methionine and valine

mitochondrial matrix

this work

Ensangp00000026391 Cofilin 16.93/7.28

2-DE-MSLC MS/MS

- 170 42 LFLMSWCPDTAK Binds actin and assists in

cytoplasmic T [9]

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iTRAQ translocation of actin from the cytoplasm to the nucleusessential for cytokinesis, endocytosisand other cell processes that require rapid turnover of actin filaments

Ensangp00000022538d

),e)Creatine kinase 26.4/5.18 2-DE-MS

ITRAQ- 90 30 AVQQQLIDDHFLFK

TFLVWCNEEDHLRPhosphorylation

cytoplasmic P [12]

Ebsangp00000020091e

)Cytochrome c 11.78/10.

17iTRAQ GDLIAYLK Electron

tranportermitochondrial membrane

this work

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Ensangp00000025174and/orEnsangp00000018280d),e),f)

D7 precursor allergen AED A2

35.57/5.7and/or32.7/5.1

1-DE-MS2-DE-MSLC MS/MS iTRAQ

42% 113-

119,

121-

125,

149-

151,

154,

169

(19-33) PSD



Ensangp00000018340*, d),e)


18.73/9.57


20% - - BLVESTSGEAFKKLPALSQYSSVVDKKVFDTVELVKCLVESTSGEAFK

Anti-inflammatory Scavenger of biogenic amines


Ensangp00000018371*,d),e)


18.46/4.8 1-DE-MS2-DE-MS LC MS/MS iTRAQ

28% 181-

183

PSD ANTFYTCFLGTSSLAGFKESVLLELLQRHMQBVLEVVGFVDGNGEVKKANTFYTCFLGTSSLAGFKMQTSDPFDMNRNAVDYNELLKQYTPVSSDDMDK



Ensangp00000018330and/orEnsangp00000025580d), e),f)


19.66/4.46and/or18.6/4.5

1-DE-MS2-DE-MSLC MS/MS ITRAQ

33% 180-

181

PSD ANTFYTCFLGTSSAQAFKAGKLDMGTTFNAGQVSALMKLDMGTTFNAGQVSALMKYAVDYVELLR



Ensangp00000018328d

), e)D7 related-4 protein precursor

19.29/7.4 1-DE-MS2-DE-MSLC MS/MS iTRAQ

30% 171-

176

22-40 LYDPLNIIELDKCIGECVQVPTSERRYEIIEGPEMDKYTAEFVQIMKVFDLMELK


secreted P [12]

Ensangp00000018321e


18.79/5.82

LC MS/MS iTRAQ

SGSFFSCMLR ? secreted P [12]

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Ensangp00000026077e

)

Disulfide isomerase precursor

55.41/4.61

iTRAQ - - - ELETVEAAEEFLKILEFFGMKILEFVQSFLDGKQTVPTCTPAPSVPRIPQIPATLSPTRVQFSCVPCPLNQRKGSVDPTLETVVPRSSVILAPEYAKAAKVLADKESNIKL

Catalyzes the rearrangement of -s-s-bonds in proteins

endoplasmic reticulum lumen

P [12]

Ensangp00000027211 Disulfide isomerase precursor

54.31/5.47

2-DE-MSiTRAQ

- 52 15 QGETDAVFLFR Catalyzes the rearrangement of -s-s-bonds in proteins


Ensangp00000002028e

)DNA helicase 117.99/8.

45iTRAQ - - - EKLIIIGDR ATP binding ? this work

Ensangp00000014287d

),e)Electron transfer flavoprotein subunit alpha mitochondrial pecursor

34.14/8.62

2-DE-MSiTRAQ

- 113 33 FTHIVAGATAFGK Participates in catalyzing the initial step of the mitochondrial fatty acid beta-oxidation


Ensangp00000018531 Enolase 28.68/9.93

iTRAQ - - - AAVPSGASTGVHEALELREALNLIQDAIAKGNPTVEVDLVTDLGLFR

Glycolytic enzyme

cytoplasmic* P [12], P[50]

Ensangp00000010297e

)Enzyme 79.47/9.6

7iTRAQ - - - LTSIPTALDLALTGK Includes

enoyl coA hydrataseMay be involved in fatty-acid metabolism


Ensangp00000021863e

)

Epilepsy holoproencephaly candidate

29.13/6.92

iTRAQ - - - YEELIRTNR oxidoreductase activity

? this work

Ensangp00000003806d

)Facilitated glucose transporter

16.83/8.48

LC MS/MS - - - HISQIVPLVAKGFSSKPLVP Sugar transporter

membranar this work

Ensangp00000000937d

)Probable Fatty acid binding protein

19.37/9.59

LC MS/MS - - - LGGGFDEETVDGR Fatty acid binding protein


Ensangp00000024159e

)Fructose biphosphate

39.18/7.72

iTRAQ - - - KPTAQEIALATVTALRIVPIVEPEILPDGDHDLER

Glycolytic enzyme

? P[12], P[50]

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aldolaseEnsangp00000020828e

)Fumarase mitochondrial precursor

50.22/7.55

iTRAQ - - - IADAIALAADDVISGK Generation of precursor metabolites and energy


Ensangp00000017396e

)Fumaryl aceto acetase

45.64/6 iTRAQ - - - GTKQVSLAGGETR Last enzyme of the tyrosine catabolic pathway


Ensangp00000029040e

)Glutathion S transferase

19.16/7.5 iTRAQ - - - LYFDMGTLYQR Key role in cellular detoxification

cytoplasmic and nuclear

this work

Ensangp00000011661e

)Glutathion S transferase (class theta)

23.78/6.51

2-DE-MS - 155 33 - this workP [50]

Ensangp00000010360e

)Glyceraldehyde phosphate dehydrogenase

35.46/8.55

iTRAQ - - - AGAEYVVESTGVFTTTEKWRDGKLTGMGCLVVNASVVAIIIPAATGHATTATQKTAFRVPTPNVSLSKPATYDQIGAAKAVGKVIP

Plays an important role in glycolysis and gluconeogenesis

cytoplasmic P[12], P[50]

Ensangp00000024265e

)Glycin cleavage system H protein mitochondrial precursor

13.52/4.2 iTRAQ - - - LMSEEQYTEFLK Catalyses the catabolism of glycine in eukaryotes


Ensangp00000010081d

)Glycogen phosphorylase

96.4/6.33 1-DE-MS 18% - - - Carbohydrate metabolism


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Ensangp0000000998e) GSG3 20.01/4.34

2-DE-MS - 75, 76 PSD - ? secreted T [51]

Ensangp00000003578e

)

GSG5 precursor 38.2/6.42 LC MS/MS iTRAQ

- - - TYFQNEFVEYR ? secreted T [51]

Ensangp00000019455d

),e)GSG6 13.05/5.1


36% - - EPLPYMYACPGTEPCQSSDRETREPLPYMYACPGTEPCQSSDRSMHDVLCDRIDQAFLEQ


Ensangp00000021970d

),e)GSG7 16.29/8.4

6LC MS/MS iTRAQ

- - - TLADETAQCMRTLADETAQCLRYGVQNQLR

? secreted P [12]

Ensangp00000005326d

)Guanine nucleotide releasing factor

137.53/9.17

LC MS/MS - - - LIEKALIYK May play a role in intracellular signaling cascade

membrane-associated

this work

Ensangp00000021028*d),e)

putative gVAG protein precursor

28.9/8.96 1-DE-MSLC MS/MS iTRAQ

43% - - DGQMDVYYFVBNYSFTNIMDRFPYAGQNIAITQFFGYRFVSSWWSEYLDARPEHVRGGPHVGCNPPSSSGGPTCQGKKYPSSYSGKPIGHFTQIASDRMPTLTWDPELASLADANARVGCSMWYWK

Allergen. Belongs to the CAP family


Ensangp00000014839 60 kDa heat shock protein mitochondrial precursor

60.77/5.28

iTRAQ - - - VEFQDALVLFSEK Protein refolding


Ensangp00000003808e

)Histone acetyltransferase GCN5

85.65/8.9 iTRAQ - - - SIPIESIPGLR Control of amino acid synthesis

nuclear* this work

Ensangp00000009655e

)Homolog 118.45/6.

27iTRAQ - - - DGKELDLVCMQK C2 domain

(cellular proteins involved in signal transduction or membrane trafficking)Cytochrome c heme-

? this work

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binding site (electron-transfer proteins)

Ensangp00000017720d

)3 Hydroxyisobutyrate dehydrogenase mitochondrial

34.31/9.27

LC MS/MS - - - VFADIVNASTGR Involved in amino acid catabolism pathway


Ensangp00000018375e

)Hypothetical 10 kDa protein

10/6.22 LC MS/MS iTRAQ

- - - LSLQLEEFAVCKAISDLQQGLFDLNHCTK

? secreted this work

Ensangp00000018379e

)Hypothetical 10.2 kDa protein

10.13/4.52

LC MS/MS iTRAQ

- - - LQQMVEDFTACR ? secreted P [12]

Ensangp00000004315e

)Hypothetical 8.8 kDa protein

8.82/4.05 LC MS/MS iTRAQ

- - - DKPDIDPVDFLVDVIK ? secreted P [12]

Ensangp00000016660d

)Isocitrate dehydrogenase

46.96/7.59

1-DE-MS 32% - - - Plays a key role in cellular defense against oxidative stress-induced damage


Ensangp00000013285e

)3 Ketoacyl coA thiolase

41.67/8.47

iTRAQ - - - AALDAAGLKPDQVDSVNIGQVLVLSSTDGAFLPRLACAGELGLDINKLNLNGAQDILVGAAHTAGTASGIASGSRITG

Involved in biosynthetic pathways such as poly beta-hydroxybutyrate synthesis or steroid biogenesis

this work

Ensangp00000010689e

)cell division Kinase

74.82/9.33

iTRAQ - - - SLDLLDSMLVLDPPGSEDLSGEEDIGSPLLPSNRDTIQNLTPSGREIKILRQAGINMMGGAGAPAG

Protein phosphorylation


Ensangp00000020132e

)Low density lipoprotein receptor

179.24/6.29

iTRAQ - - - DGTERVLIVSQNLGSQRVELITKIVTAEIQAPDGSPDDAPADHVCACPQGLMLLK

Lipid metabolism

membranar this work

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GRTNEnsangp00000020384e

)Low density lipoprotein receptor

17.3/5.04 LC MS/MS - - - BISRAGICDGK Lipid metabolism

membranar this workP [50]

Ensangp00000022875e

)Lysozyme precursor

15.33/8.56

LC MS/MS iTRAQ

- - - NGSTDYGIFQINNKYWBDSGYGSNDCKNLLNDDITDDIKKLPNVSSCF



Ensangp00000011006e

)Malate dehydrogenase

35.37/6.95

2-DE-MS 96 PSD DDLFNTNASIVR Participates in the citric acid cycle


Ensangp00000020184d

),e)Malate dehydrogenase

35.27/9.52

LC MS/MS - - - ANTFVGEAAGVDPQK Metabolic enzymes which catalyse the last step in anaerobic glycolysis


Ensangp00000017682d

),e)Maltase 67.21/5.8


27% 6-8, 12 (17-43) AMPSGAIANWVLGNHDNSRDQPETYDMVHQWRELNVAAQLAAPRGITQTIDYLK

Sugar digestionConverts sucrose in nectar to glucose and fructose

secreted P [12], T [9]

Ensangp00000015067 Mitochondrial carrier

2-DE-MS 16 186

Ensangp00000004215e

)Mitogen activated kinase kinase kinase kinase

159.64/10.04

LC MS/MS - - - NIATYYGAFIK Protein kinaseATP binding


Ensangp00000003748e

)Myosin 121.89/10

.23iTRAQ - - - Contractile

proteincytoplasmic this work

P [52]Ensangp00000003978e

)N acylneuraminate cytidyltransferase

21.1/5.67 LC MS/MS - - - HLTLARILLGME Forms CMP-NeuAc, the nucleotide sugar donor used by sialyltransferases (modification may be important inpathogenesis)


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Ensangp00000021120e

)NADPH dependent carbonyl reductase

27.05/7.73

LC MS/MS - - - MDFTGKVVLITGASSGIGASTAK

Carbohydrate metabolism


Ensangp0000002613e) Nucleolar RNA associated protein

117.56/7.05

iTRAQ - - - LSSETIDELEK Appears to be associated with ribosome biogenesis


Ensangp00000011253d

),e)Nucleoside diphosphate kinase

19.01/8.46

1-DE-MSiTRAQ

32% - - GDLCVQVGR Maintenance of cellular pool of nucleoside triphosphates

cytoplasmic and plasma membrane

this workT [9]

Ensangp00000012716*,d),e)

Putative 5’ nucleotidase precursor

63.47/7.01


20% 10, 13-33, 38-51, 54, 57, 60, 64, 65, 77-82, 85-87,130-134, 140-141, 144-145

(15-30) PSD

APFPLTLIHINDLHARDQIYYVVVPSYLADGKDGFAMKECIAGIARGLAPYLAELEKLGTQVIGTTEVFLDRESCRLSGADLWSAIDHSFTLDDEFRMKIPTVVANLEKNVNIIVVLSHCGLDGDKQLAEEAGDLIDVIVGAHSHSLLLNKYDTIEGDYPLVVKKVVIENHTNGTCSWDLDSQRNPIEKGDITNGLAIEAAPYGSSVDMIK


Ensangp00000020778 Peptidyl prolyl cis trans isomerase

18.29/8.97

LC MS/MS iTRAQ

- - - FFDMTVDNQPLGRIVIELRPDVVPKHVVFGSVVEGMDVVR

Accelerates protein folding

cytopasmic this work

Ensangp00000028058d

),e)Peroxidase precursor

24.99/8.23


16% - - AFAGAININDHMFNPTVLERCFAIPVRPDDPVLSAGGIQCLDLVRLLPAEYGDGVYVPRSNITPELTILHVAFLRTTLVNMQFGQLVAHDMGLRWEDFVELR

Vasodilatator secreted P [12], P [50]

Ensangp00000020634e

)Peroxysomal targeting signal 2 receptor

36.41/6.22

iTRAQ - - - VSGSGDGSVQLWNTFTTNRTNLASSVQLWNTNLASN

Family of potein implicated in a variety of

? this work

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SQFYGLAGGGT functions ranging from signal transduction and transcription regulation to cell cycle control and apoptosis

Ensangp00000024749e

)Pheromone/general odorant binding protein OBP56

27.15/5.52

iTRAQ - - - SASEVQDDKCK ? ? this work

Ensangp00000013993e

)Phosphatidylethanolamine-binding protein

24.17/6.67

iTRAQ - - - YVFLVYK Proteinase inhibitor

? this workP [52]

Ensangp00000012460d

)Phosphoglycerate kinase

43.84/7.54

2-DE-MS - 109 27 - Glycolysis cytoplasmic this work

Ensangp00000015800d

),e)Phosphoglycerate mutase

28.7/6.8 LC MS/MS - 148 25 YGEEQVLIWR Involved in energy pathways


Ensangp00000020531e

)Precursor 200.9/4.5 iTRAQ - - - ERTGEIMLLQR

AGTIVGNVSALDEDVGPNGTRDARLDRDTNPESYAIGTIFVNSTLNYNYAAVIVERQLDYEEVSGVLDRFTVEMQERLANANLELS

Cadherin membranar this work

Ensangp00000012492d

)Precursor 28.47/5.0

41-DE-MS 22% - - - EGF-like

domain? this work

Ensangp00000013568d

)Precursor 41.83/5.4 2-DE-MS - 75 - - Aspartic

protease A1secreted this work

Ensangp00000016366d


32-DE-MS - 142 23 - Glucose-

methanol-choline oxidoreductase Involved in energy pathways


Ensangp00000019046d


5LC MS/MS - - - ANDRAMVK EGF-like

domain? this work

Ensangp00000029324e Precursor 25.94/4.8 LC MS/MS - - - TLTFVLKPTK Alpha 2 intracellular this work

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) macroglobulin domain

Ensangp00000031578e


8iTRAQ - - - DMPNITLLNLDGNQLSR

NLLQNLDLALFVAMPQLLNLNASSPVANNLTSAPIAPVTGRPNITLLNVSAPIGLNKITTFNIT

Leucine rich repeatPutatively involved in protein-protein interaction

? this work

Ensangp00000020734d

)Pterin 4 alpha carbinol amine dehydratase

21.20/10.23

LC MS/MS - - - LAQFLDQAAAVAK Transcriptional activator/pterin dehydratase

? this work

Ensangp00000027538c

)Retrovirus related pol polyprotein

9.51/11.28

2-DE-MS - 181, 183

PSD - ? nuclear this work

Ensangp00000021077d

),e)Ribonuclease 14.41/8.0

4LC MS/MSiTRAQ

ALAPYNQAIVADR Inhibits protein synthesis by cleavage of mRNA

? this work

Ensangp00000006850e

)DNA directed RNA polymerase

68.25/8.18

iTRAQ - - - LSYISALGMMTR Transcription nuclear this work

Ensangp00000019607e

)Ryanodine receptor 1

577.53/5.18

LC MS/MS - - - YFDMFLKLK Ca2+ release channels involved in secretory pathways ?

membranar this work

Ensangp00000020530e

)Serine protease precursor

25.2/4.57 iTRAQ NGQNDIALLQLDRKVITSAQCTTDEGNGIPSVVRLGGTKSVLFAVLLIWDSVVALLQLDRKIIINTTDEGNGIPSVVR

Involved in immunity or in coagulation cascade

secreted this work

Ensangp00000016680 Serpin 9 46.36/7 iTRAQ - - - LAAETDILHEVVNEGISR Serine protease inhibitor Involved in immunity

secreted T [11]

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Ensangp00000017327 putative Salivary protein SG1B

46.6/7.37 iTRAQ - - - DYESYLGAMFAADAFHVVYEADGK

? secreted P [12]

Ensangp00000032098e

),f)Salivary D3 protein iTRAQ - - - AAAGPAPDPSSQFCQQLLDDA

QRSaglin ? P [12]

Ensangp00000027418d

),*Salivary gland 1-like 3 protein

44.51/6.04

1-DE-MS 30% - - - ? secreted P [12]

Ensangp00000009988e

)SG3 20.01/4.3 iTRAQ ATGPLFLPHFGQGPR

RGQQLIFLAASVERNPAATIAVASAATASPTTAEAQQQRQQVQR

Mucin secreted T [51]

Ensangp00000008103e

)Stromal interaction molecule precursor

54.49/6.36

LC MS/MS - - - DVEGLLKAEVALK Role in RNA binding

membranar this work

Ensangp00000009009e

)Fact complex subunit facilitates chromatin transcription

71.65/6.28

iTRAQ - - - RPLSAYMLWLNSAR Recombination signal sequence recognition T160

nuclear this work

Ensangp00000016164e

)Superoxyde dismutase

15.67/5.45

iTRAQ - - - SLVVHADPDDLGVGGHELSK Metalloprotein that prevents damage by oxygen-mediated free radicals


Ensangp00000018041d

)Toll precursor 16.69/4.5

12-DE-MS - 152 17 - Toll IA

Involved in signal transduction pathways in response to pathogens

plasma membrane

this work P [50]

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Ensangp00000028309e

)

Trans enoyl COA isomerase mitochondrial precursor

30.18/7.13

LC MS/MS - - - ALEQAVAFLNR Fatty acid metabolism


Ensangp00000021085e

)

Translationally controlled tumor protein TCTP

19.54/4.42

iTRAQ - - - LVDDVMYEVYGK Histamine-releasing factor

? T [9], T[11]

Ensangp00000017522e

)Trio protein 43.78/6.4 iTRAQ - - - SMYDLIGQLVQSSK ? secreted P [12]

Ensangp00000018152d

),e)Triosephosphate isomerase

22.52/5.09

1-DE-MSLC MS/MS

30% - - AIFGETDELIAEKDWSNVVIAYEPVWAIGTGKSLLPETIGVAAQNCYKDLGLGWVILGHSER

Central enzyme in the glycolytic pathwayPlays an important role in several metabolic pathways

cytoplasmic this workP [52]

Ensangp00000025045e

)Trypsin precusror 28.65/6.4

9iTRAQ QIGIVSWGDTQCVGT

RGGSSTLNETDLTVRRLALTAGHNGNFVPNLPAPARATGRIV

Proteolytic enzyme

secreted this work

Ensangp00000008105e

)E3 Ubiquitin ligase 201.24/6.

46iTRAQ GLAMADLDRLEK

QQLCIKPNPDNSEHRNHKGTYHSVNTQASQQQQAPLLRDGSRVMMMG

Involved in protein degradation pathway


Ensangp00000000334c

)e)Unknown 39.57/7.2

9LC MS/MS - - - SPILLLDDIFDK ATP/GTP-

binding site motif A (P-loop)


Ensangp00000012072d

)Unknown 29.2/4.4 2-DE-MS - 135 20 DSTLIMQLLR 14-3-3

protein. Family of conserved regulatory molecules

cytoplasmic P [12]

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that bind a multitude of functionally diverse signaling proteins

Ensangp00000012822e

)Unknown 74.9/7.88 iTRAQ DVQASHISRLGTSSIVSYTP

TLRNGTPQASNSIYCTLRNGTNVSMCPDTIDSD

Immunoglobulin-like domainInvolved in cell adhesion

membrane this work

Ensangp00000015472c

)e)Unknown 15.64/10.

381-DE-MS 20% - - - InterPro

Zn-finger, C2H2 typenucleic acid-binding protein

nuclear ? this workP [50], T [9]

Ensangp00000016832e

)Unknown 19.42/4.8

8iTRAQ QQAAAAAETTSQAAGTLMDH

AKAnti-freeze protein

? this work

Ensangp00000017135e

)Unknown 85.43/8.6

4LC MS/MS iTRAQ

- - - IKCGLLLEGVR ? ? this work

Ensangp00000019537e

)Unknown 90.81/7.4

1iTRAQ - - - ? ? this work

Ensangp00000019887d

)Unknown 70.9/5.1 2-DE-MS - 9 18 - Heat-shock

70 domainMay be involved in response to stress

cytoplasmic and organelles

P [12]

Ensangp00000028177e

)Unknown 36.81/10.

03iTRAQ - - - LGIGSSSINGSGAVVRK Basic helix-

loop-helix dimerisation region

this work

Ensangp00000028294d

)Unknown 15.18/4.5

7LC MS/MS - - - GSTINLTBAVK Immunoglob

ulin-like domainInvolved in cell adhesion

membrane ? this work

Ensangp00000029447e

)Unknown 20.35/6.2

4iTRAQ - - - EQQQLALDVR ? secreted this work

Ensangp0000012893e) Unknown 72.74/4.92

iTRAQ - - - ELEDIVQPIIAK Hsp70 and tropomyosin domains

ER ? this work

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Ensangp00000011593e

)Wilm’s tumor 1 associating WT1 associated pr splicing regulator female lethal 2-D homolog

33.55/4.78

LC MS/MS - - - FTPDSNTGKR Potential role in transcriptional regulationInvolves in alternative splicing regulation

nuclear this work

a) When several spots corresponded to the same protein, the percentage range of the sequence coverage is indicated in parenthesis. b) Subcellular localization is

inferred from sequence or structure similarity with orthologous proteins. c) Identification was performed using Ensembl database v35 of november 2005. d)

proteins identified from salivary gland extracts of young blood-fed females. e) Proteins identified from salivary gland extracts of olf blood-fed females. f)

Proteins allowing a correction of incorrect genome annotation (the part of the sequence in bold is that described in Ensembl v43. PSD : post source decay.

Shaded lines : Proteins identified for the first time by a proteomic approach. * means that the proteins were also identified in saliva. References are underlined

when they correspond to proteins identified in human saliva.

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Salivary components were separated by a 12% NU-PAGE Bis-Tris gel under denaturating and reducing conditions. Molecular mass markers are shown on the left. After Coomassie staining, the gel was cut into millimeter slices as indicated by the numbers on the right side of the figure. The plugs obtained were analyzed by mass spectrometry as described

in the Methods section.

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Saliva was collected from 7200 females using artificial feeders. After lyophilisation, saliva components were re-suspended in water and aliquots were analyzed by SDS-PAGE.

Following silver nitrate staining, the numbered protein bands were analyzed by mass spectrometry

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For Peer Reviewunknown35%

signal transduction5%

blood sugar feeding22%

protein folding

modification degradation

cytokinesis

endocytosis

energy metabolism 12%

sugar metabolism3%

protein synthesis3%

lipid metabolism5%

immune response and defense

8%

B

DNA replication7%

signal transduction3%

cytokinesis

endocytosis

energy metabolism16%


amino acid metabolism

3%

protein synthesis3%

unknown33%

sugar metabolism3%

A

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amino acid metabolism6%

unknown36%

stress and ageing response

3% signalling3%

cellular detoxification2%

DNA replication recombination

3%

energy metabolism16%

cytokinesis endocytosis exocytosis

3%

lipid metabolism3%

carbohydrate

metabolism

transcription regulation3%

response to pathogens3%


protein folding modification degradation

7%protein synthesis2%

2-DE-MS25%

1-DE-MS12%iTRAQ

19%

LC MS-MS44%

A

B

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